CUT&RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&RUN.
Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin Immunoprecipitation (ChIP) has been the gold standard for determining protein localization for over 30 years and is defined by the use of an antibody to pull out the protein of interest from sonicated or enzymatically digested chromatin. More recently, antibody tethering techniques have become popular for assessing protein localization on chromatin due to their increased sensitivity. Cleavage Under Targets & Release Under Nuclease (CUT&RUN) is the genome-wide derivative of Chromatin Immunocleavage (ChIC) and utilizes recombinant Protein A tethered to micrococcal nuclease (pA-MNase) to identify the IgG constant region of the antibody targeting a protein of interest, therefore enabling site-specific cleavage of the DNA flanking the protein of interest. CUT&RUN can be used to profile histone modifications, transcription factors, and other chromatin-binding proteins such as nucleosome remodeling factors. Importantly, CUT&RUN can be used to assess the localization of either euchromatic- or heterochromatic-associated proteins and histone modifications. For these reasons, CUT&RUN is a powerful method for determining the binding profiles of a wide range of proteins. Recently, CUT&RUN has been optimized for transcription factor profiling in low populations of cells and single cells and the optimized protocol has been termed ultra-low input CUT&RUN (uliCUT&RUN). Here, a detailed protocol is presented for single-cell factor profiling using uliCUT&RUN in a manual 96-well format.
Many nuclear proteins function by interacting with chromatin to promote or prevent DNA-templated activities. To determine the function of these chromatin-interacting proteins, it is important to identify the genomic locations at which these proteins are bound. Since its development in 1985, Chromatin Immunoprecipitation (ChIP) has been the gold standard for identifying where a protein binds to chromatin1,2. The traditional ChIP technique has the following basic workflow: cells are harvested and crosslinked (usually with formaldehyde), chromatin is sheared (usually with harsh sonication methods, necessitating crosslinking), the protein of interest is immunoprecipitated using an antibody that targets the protein (or tagged protein) followed by a secondary antibody (coupled to agarose or magnetic beads), crosslinking is reversed, protein and RNA are digested to purify DNA, and this ChIP enriched-DNA is used as the template for analysis (using radiolabeled probes1,2, qPCR3, microarrays5,6, or sequencing4). With the advent of microarrays and massively parallel deep sequencing, ChIP-chip5,6 and ChIP-seq4 have more recently been developed and allow for genome-wide identification of protein localization on chromatin. Crosslinking ChIP has been a powerful and reliable technique since its advent with major advances in resolution by ChIP-exo7 and ChIP-nexus8. In parallel to the development of ChIP-seq, native (non-crosslinking) protocols for ChIP (N-ChIP) have been established, which utilize nuclease digestion (often using micrococcal nuclease or MNase) to fragment the chromatin, as opposed to sonication performed in traditional crosslinking ChIP techniques9. However, one major drawback to both crosslinking ChIP and N-ChIP technologies has been the requirement for high cell numbers due to low DNA yield following the experimental manipulation. Therefore, in more recent years, many efforts have been toward optimizing ChIP technologies for low cell input. These efforts have resulted in the development of many powerful ChIP-based technologies that vary in their applicability and input requirements10,11,12,13,14,15,16,17,18. However, single-cell ChIP-seq based technologies have been lacking, especially for non-histone proteins.
In 2004, an alternative technology was developed to determine protein occupancy on chromatin termed Chromatin Endogenous Cleavage (ChEC) and Chromatin Immunocleavage (ChIC)19. These single-locus techniques utilize a fusion of MNase to either the protein of interest (ChEC) or to protein A (ChIC) for direct cutting of DNA adjacent to the protein of interest. In more recent years, both ChEC and ChIC have been optimized for genome-wide protein profiling on chromatin (ChEC-seq and CUT&RUN, respectively)20,21. While ChEC-seq is a powerful technique for determining factor localization, it requires developing MNase-fusion proteins for each target, whereas ChIC and its genome-wide variation, CUT&RUN, rely on an antibody directed toward the protein of interest (as with ChIP) and recombinant Protein A-MNase, where the Protein A can recognize the IgG constant region of the antibody. As an alternative, a fusion Protein A/Protein G-MNase (pA/G-MNase) has been developed that can recognize a broader range of antibody constant regions22. CUT&RUN has rapidly become a popular alternative to ChIP-seq for determining protein localization on chromatin genome-wide.
Ultra-low input CUT&RUN (uliCUT&RUN), a variation of CUT&RUN that enables the use of low and single-cell inputs, was described in 201923. Here, the methodology for a manual 96-well format single-cell application is described. It is important to note that since the development of uliCUT&RUN, two alternatives for histone profiling, CUT&Tag and iACT-seq have been developed, providing robust and highly parallel profiling of histone proteins24,25. Furthermore, scCUT&Tag has been optimized for profiling multiple factors in a single cell (multiCUT&Tag) and for application to non-histone proteins26. Together, CUT&RUN provides an attractive alternative to low input ChIP-seq where uliCUT&RUN can be performed in any molecular biology lab that has access to a cell sorter and standard equipment.
Ethics statement: All studies were approved by the Institutional Biosafety Office of Research Protections at the University of Pittsburgh.
1. Prepare magnetic beads
NOTE: Perform prior to cell sorting and hold on ice until use.
2. Harvest cells
NOTE: This step is written for adhered cells and optimized for murine E14 embryonic stem cells. Culturing and harvesting the cells depend on the cell type.
3. Cell sorting and lysis
4. Pre-block samples to prevent early digestion by MNase
5. Addition of primary antibody
6. Addition of pA-MNase or pA/G-MNase
NOTE: Protein A has a high affinity for IgG molecules from certain species such as rabbits but is not suitable for IgGs from other species such as mice or rats. Alternatively, Protein A/G-MNase can be used. This hybrid binds rabbit, mouse, and rat IgGs, avoiding the need for secondary antibodies when mouse or rat primary antibodies are used.
7. Directed DNA digestion
8. Sample fractionation
9. DNA extraction
10. End repair, phosphorylation, adenylation
NOTE: The reagents are sourced as referenced in the Table of Materials. The below protocol follows a similar method to the commercial kit such as NEBNext Ultra DNA II kit.
11. Adapter ligation
NOTE: Keep the samples on ice while setting up the following reaction. Allow ligase buffer to come to room temperature before pipetting. Dilute the Adaptor (see Table of Materials) in a solution of 10 mM Tris-HCl containing 10 mM NaCl (pH 7.5). Due to the low yield, do not pre-quantify the CUT&RUN-enriched DNA. Rather, generate 25-fold dilutions of the adaptor, using a final working adaptor concentration of 0.6 µM.
12. USER digestion
13. Polystyrene-magnetite bead clean-up following ligation reaction
NOTE: Allow polystyrene-magnetite beads to equilibrate at room temperature (~30 min). Vortex to homogenize the bead solution before using. Perform the following steps at room temperature.
14. Library enrichment
NOTE: Primers are diluted with the same solution as the adaptor. For this library build, use a final working primer concentration of 0.6 µM.
15. Polystyrene-magnetite bead clean-up
Here, a detailed protocol is presented for single-cell protein profiling on chromatin using a 96-well manual format uliCUT&RUN. While results will vary based on the protein being profiled (due to protein abundance and antibody quality), cell type, and other contributing factors, anticipated results for this technique are discussed here. Cell quality (cell appearance and percent of viable cells) and single-cell sorting should be assessed prior to or at the time of live-cell sorting into the NE buffer. An example of ES cell colonies and cell sorting is shown in Figure 2A,B. Specifically, low-quality cells should not be used, and if the quality is an issue, care should be taken to follow guidelines for the specific cell type. In addition, accurate cell sorting by the cell sorting instrument should be assessed in advance of experimentation. For example, test cells could be sorted and stained using Hoechst 33342 stain and counted to assure either 0 or 1 cell is found in each well. If single cells are not found, sorting conditions must be optimized. After library preparation, samples can be assessed on either an agarose gel (if the concentration permits for loading of 30 ng or more, as there is a lower limit to DNA visualization on an agarose gel) or a Fragment Analyzer (or similar device such as a Tapestation or Bioanalyzer) prior to sequencing and example results are shown in Figure 2C,D. Specifically, the expected size distribution is from ~150 to ~500 bp. In higher cell amounts, CUT&RUN performed on large proteins (such as histones) will have a right-sided size distribution, where the majority of DNA will be seen ~270 bp; however, this shoulder is typically not observed in single-cell experiments.
After sequencing, the quality of the sequencing reads should be assessed using FASTQC. The percent of uniquely mapping reads should be determined. Typically, 0.5%-10% uniquely mapping reads are observed in single-cell experiments. These mapping percentages are similar to other DNA-based single-cell techniques30. Next, the size distribution of the reads after mapping should be determined to assure the profile is similar to pre-sequencing (with the adaptor sequence no longer contributing to the read sizes).
After data quality has been assessed, protein occupancy can be visualized using various methods: single locus genome browser images can be visualized using UCSC genome browser or IGV (Figure 3A) and genome-wide occupancy patterns over specific genomic coordinates can be visualized using metaplots (Figure 3B, bottom), heatmaps (Figure 3B, top), or 1D heatmaps (Figure 3C). For more information on data analysis, refer to the study by Patty and Hainer27. Single-cell data from a diploid cell will result in up to four reads contributing to each locus (four reads if the cell was in mitosis), but more often one or two reads. Therefore, the data is binary, and a high background can be more easily mistaken for occupancy, relative to high cell experiments. Therefore, it is recommended to perform a parallel CUT&RUN experiment on a high cell number (5,000 to 100,000 cells), if possible, to acquire all the possible binding locations for the protein of interest. Then, single-cell data can be compared to possible binding locations. In the examples shown in Figure 3, single-cell CTCF uliCUT&RUN results are compared to high cell CTCF uliCUT&RUN (Figure 3A) or ChIP-seq (Figure 3B,C). As demonstrated previously, CTCF, SOX2, and NANOG single-cell uliCUT&RUN peaks largely represented stronger peaks from high cell ChIP-seq datasets23.
Figure 1: Schematic of the uliCUT&RUN protocol. Cells are harvested and sorted into a 96-well plate containing NE buffer. Individual nuclei are then bound to ConA-conjugated paramagnetic microspheres, and sequentially an antibody (targeting the protein of interest) and pA-MNase or pA/G-MNase are added. Protein-adjacent DNA is cleaved via MNase, and DNA is then purified for use in library preparation. This figure was created with Biorender.com. Please click here to view a larger version of this figure.
Figure 2: Example results from cell sorting and quality control of uliCUT&RUN libraries. (A) Image of high-quality murine embryonic stem (ES) cells. Scale bar: 200 µm. (B) Output from single-cell sorting after adding 7AAD to harvested ES cells and sorting on a FACS instrument. (C) Ethidium bromide-stained agarose gel depicting completed uliCUT&RUN libraries. Lane 1 is a low molecular weight ladder and lanes 2-21 are examples of successful individual single-cell uliCUT&RUN libraries prior to sequencing. (D) Ethidium bromide-stained agarose gel depicting sub-optimal and optimal completed uliCUT&RUN libraries. Lane 1 is a low molecular weight ladder, lane 2 is a sub-optimal library due to inefficient MNase digestion, and lane 3 is a successful library with appropriate digestion. (E) Fragment analyzer distribution of one single cell uliCUT&RUN library prior to sequencing. Please click here to view a larger version of this figure.
Figure 3: Example of expected results for single ES cell uliCUT&RUN data. (A) Browser track of high cell number (5,000 cells) CTCF or negative control (No Antibody, No Ab) uliCUT&RUN (top two tracks) and single-cell CTCF uliCUT&RUN. The image is reproduced, with permission, from Patty and Hainer27. (B) Heatmaps (top) and metaplots (bottom) of single-cell CTCF or negative control (No Ab) uliCUT&RUN over previously published CTCF ChIP-seq sites (GSE11724). The image is reproduced, with permission, from Hainer et al.23. (C) 1D heatmaps of single-cell CTCF or negative control (No Ab) uliCUT&RUN over previously published CTCF ChIP-seq sites (GSE11724). The image is reproduced, with permission, from Hainer et al.23. Please click here to view a larger version of this figure.
Table 1: Composition of various buffers used in this protocol. Volume of stock solution required is listed with final concentration written in parentheses. Please click here to download this Table.
CUT&RUN is an effective protocol to determine protein localization on chromatin. It has many advantages relative to other protocols, including: 1) high signal-to-noise ratio, 2) rapid protocol, and 3) low sequencing read coverage required thus leading to cost savings. The use of Protein A- or Protein A/G-MNase enables CUT&RUN to be applied with any available antibody; therefore, it has the potential to quickly and easily profile many proteins. However, adaptation to single-cell for any protein profiling on chromatin has been difficult, especially when compared to single-cell transcriptomics (i.e., scRNA-seq), due to the low copy number of DNA relative to RNA (two to four copies of DNA versus possible thousands of RNA copies).
In the protocol detailed above, several critical steps should be considered. First, the appropriate sorting of cells is dependent on the cell (or tissue) type, and care should be taken for accurate sorting. It is recommended to test how effective single-cell sorting is with the instrument in advance of any experiment. Second, effective antibody choice is essential. Before proceeding to a single-cell application, it is recommended to test the antibody in a high cell number experiment (as well as other standard antibody tests, such as confirming specificity using western blot after knockdown, titration of the antibody in CUT&RUN experiments, etc.). Third, use a negative control, such as IgG or no primary antibody, because an appropriate comparison to the experimental samples is essential for the interpretation of the data quality and biological results. When comparing single-cell experimental results to a high cell number dataset, the negative control single-cell experiments should have less read coverage over those binding sites identified in the high cell experiment and rather have a random distribution of reads across the genome (with a bias for open regions of chromatin). Fourth, care should be taken when adding and activating the Protein A- or Protein A/G-MNase so as not to over digest the chromatin: do not overheat the samples with your hands, maintain the samples in an ice/water-bath temperature (0 °C), and chelate the reaction at the appropriate time. Fifth, care should be taken throughout the uliCUT&RUN experiment and library preparation, due to low material. For example, extended incubation on the magnetic rack during bead binding to assure the supernatant has cleared and taking care not to disturb the beads when removing the supernatant are essential for sufficient yield. Sixth, depending on the questions being addressed, the number of single-cell experiments being performed is an important consideration. Some of the single-cell experiments will fail (as with all low-input experiments), and, therefore, the number of positive experimental results required for appropriate interpretation should be considered in advance of beginning the experiment. The number of cells to include in the experiment is dependent on the amount of experimental data required by the investigator and the quality of the antibody. Finally, note that equivalent results could be achieved with reduced volumes of many aspects of this protocol. Steps where the volume was reduced by 50% include the volume of NE buffer, wash buffer, primary antibody mixture (including the amount of primary antibody), pA-MNase mixture (including the amount of pA-MNase), and all steps in the library preparation.
Based on the complicated nature of factor profiling on chromatin, there are many potential sources of issues and places where troubleshooting may become necessary. While there may be many steps where issues can arise, three major issues have been observed: 1) low DNA yield for input into library build; 2) high background signal in experimental samples, and 3) low yield after library build. If there is insufficient DNA for library preparation and sequencing (point 1), note the following troubleshooting advice: a) there may have been incomplete membrane lysis and therefore the lysis time with NE buffer can be increased; b) there may have been inefficient binding of the nucleus to the ConA-conjugated paramagnetic microspheres and this could be remedied through appropriate mixing upon addition of these beads; c) there may have been too little antibody added, and therefore it is recommended to perform a titration of antibody to identify the most effective amount; d) incubation times with either the primary antibody or the Protein A- or Protein A/G-MNase are either too short (i.e., not enough time to permit binding) or too long (these are native, uncrosslinked samples), and could be optimized; e) the interaction of the target protein with chromatin could be too transient to capture in native conditions and therefore crosslinking CUT&RUN could be performed28. While single-cell datasets will yield high background, there may be excessively high background where the signal is hard to interpret from the background (point 2). For this issue, note the following advice: a) the blocking step with EDTA to prevent pre-emptive MNase digestion could be increased or optimized; b) if there is excessive cutting, it could be due to having too much Protein A- or Protein A/G-MNase, and therefore a titration of appropriate amounts can be performed; c) over digestion by MNase could result in the high background, and therefore the appropriate mixing of calcium chloride upon addition and optimization for MNase digestion time should be assessed. Finally, efficient uliCUT&RUN-enriched DNA may have been recovered, but a low amount of library may be recovered (point 3). For this issue, the following are recommended: a) appropriate handling and use of polystyrene-magnetite beads to assure the correct purification and no DNA loss; specifically, it is recommended to have 15 min incubations and a minimum of 5 min to magnetize beads to prevent loss; b) under-amplification of the library at the PCR stage would result in low yield and therefore the appropriate cycles should be determined using qPCR (as previously established for ATAC-seq libraries29).
As with all technologies, there are limitations to uliCUT&RUN that should be considered before initiating any experimentation. First, these experiments are designed and optimized for native conditions, and therefore if a protein is only transiently interacting with chromatin, a crosslinking approach may be necessary to ensure recovery of the interaction. Second, as with all antibody-based techniques, the quality of the antibody is important. Care should be taken to assure the quality and consistency of the antibody in advance of undertaking any experiments. Third, MNase background cutting can occur and, while there is a consistently lower background signal in CUT&RUN relative to other experiments, the background signal can be high in single-cell experiments and therefore appropriate controls and analyses should be performed. While the binary nature of single-cell profiling data limits the visualization, more advanced computational genomic technologies, such as dimensional reduction and others can be performed (as previously described27). Finally, while single-cell profiling has been expanded here to 96-well format, this is low throughput relative to other single-cell technologies that utilize 10xGenomics or other formats.
Tethering-based profiling technologies such as ChEC-seq20, CUT&Tag24, CUT&RUN21, and uliCUT&RUN23, can determine factor localization on chromatin with a faster experimental timeline, lower background, and lower cost than traditional profiling technologies, such as ChIP-seq. Therefore, these are very exciting technologies for application to precious samples such as patient samples or early developmental samples. Furthermore, application to single cells can provide complementary studies performed using other single-cell experiments such as scRNA-seq and scATAC-seq30. As described using these more broadly used single-cell technologies, novel insights can be gained relative to bulk cell experiments. Single-cell protein profiling on chromatin is anticipated to become more regularly used as the technologies continue to improve and permit more parallelization.
The authors have nothing to disclose.
We thank members of the Hainer Lab for reading and comments on an earlier version of this manuscript. This project used the NextSeq500 available at the University of Pittsburgh Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh for sequencing with special thanks to its director, William MacDonald. This research was supported in part by the University of Pittsburgh Center for Research Computing through the computer resources provided. This work was supported by the National Institutes of Health Grant Number R35GM133732 (to S.J.H.).
1.5 mL clear microfuge tubes | ThermoFisher Scientific | 90410 | |
1.5 mL tube magnetic rack | ThermoFisher Scientific | 12321D | |
1.5 mL tube-compatible cold centrifuge | Eppendorf | 5404000537 | |
10 cm sterile tissue culture plates | ThermoFisher Scientific | 150464 | |
10X T4 DNA Ligase buffer | New England Biolabs | B0202S | |
15 mL conical tubes | VWR | 89039-656 | |
1X TE buffer | ThermoFisher Scientific | 12090015 | |
200 µL PCR tubes | Eppendorf | 951010022 | |
2X quick ligase buffer | New England Biolabs | M2200 | Ligase Buffer |
5X KAPA HiFi buffer | Roche | 7958889001 | 5X high fidelity PCR buffer |
7-Amino-Actinomycin D (7-AAD) | Fisher Scientific | BDB559925 | |
96-well magnetic rack | ThermoFisher Scientific | 12027 or 12331D | |
96-well plate | VWR | 82006-636 | |
AMPure XP beads | Beckman Coulter | A63881 | polystyrene-magnetite beads; Due to potential variability between AMPure XP bead lots, it is recommended that your AMPure bead solution be calibrated. See manufacturer’s instructions |
Antibody to protein of interest | varies | ||
ATP | ThermoFisher Scientific | R0441 | |
BioMag Plus Concanavalin A beads | Polysciences | 86057-10 | ConA-conjugated paramagnetic microspheres |
BSA | |||
Calcium Chloride (CaCl2) | Fisher Scientific | AAJ62905AP | |
Cell sorter | BD FACSAria II cell sorter | Requires training | |
Cell-specific media for cell culture | Varies | ||
Chloroform | ThermoFisher Scientific | C298-500 | Chloroform is a skin irritant and harmful if swallowed; handle in a chemical fume hood using gloves, a lab coat, and goggles |
Computer with 64-bit processer and access to a super computing cluster | For computational analyses of resulting sequencing datasets | ||
DNA spin columns | Epoch Life Sciences | 1920-250 | |
dNTP set | New England Biolabs | N0446S | |
EGTA | Sigma Aldrich | E3889 | |
Electrophoresis equipment | varies | ||
Ethanol | Fisher Scientific | 22032601 | 100% vol/vol ethanol is highly flammable; handle in a chemical fume hood using gloves, a lab coat, and goggles |
Ethylenediaminetetraacetic acid (EDTA) | Fisher Scientific | BP2482100 | |
FBS | Sigma Aldrich | F2442 | |
Glycerol | Fisher Scientific | BP229-1 | |
Glycogen | VWR | 97063-256 | |
HEPES | Fisher Scientific | BP310-500 | |
Heterologous S. cerevisiae DNA spike-in | homemade | Prepared from crosslinked, MNase-digested, and agarose gel extracted genomic DNA purified of protein/RNA and diluted to 10 ng/mL. We recommend yeast genomic DNA, but other organisms can be used if needed. | |
Hydrochloric Acid (HCl) | Fisher Scientific | A144-212 | Hydrochloric Acid is very corrosive; handle in a chemical fume hood using gloves, a lab coat, and goggles |
Ice Bucket | varies | ||
Illumina Sequencing platform (e.g., NextSeq500) | Illumina | ||
Incubator with temperature and atmosphere control | ThermoFisher Scientific | 51030284 | |
KAPA HotStart HiFi DNA Polymerase with 5X KAPA HiFi buffer | Roche | 7958889001 | hotstart high fidelity polymerase |
Laminar flow hood | Bakery Company | SG404 | |
Manganese Chloride (MnCl2) | Sigma Aldrich | 244589 | |
Micropipette set | Rainin | 30386597 | |
Minifuge | Benchmark Scientific | C1012 | |
NEB Adaptor | New England Biolabs | E6612AVIAL | Adaptor |
NEB Universal primer | New England Biolabs | E6611AVIAL | Universal Primer |
NEBNext Multiplex Oligos for Illumina kit | New England Biolabs | E7335S/L, E7500S/L, E7710S/L, E7730S/L | Indexed Primers |
Negative control antibody | Antibodies-Online | ABIN101961 | |
Nuclease Free water | New England Biolabs | B1500S | |
PCR thermocycler | Eppendorf | 2231000666 | |
Phase lock tubes | Qiagen | 129046 | |
Phenol/Chloroform/Isoamyl Alcohol (PCI) | ThermoFisher Scientific | 15593049 | Phenol is harmful if swallowed or upon skin contact; handle in a chemical fume hood using gloves, a lab coat, and goggles |
Phsophate buffered saline (PBS) | ThermoFisher Scientific | 10814010 | |
Pipette aid | Drummond Scientific | # 4-000-100 | |
Polyethylene glycol (PEG) 4000 | VWR | A16151 | |
Potassium Chloride (KCl) | Sigma Aldrich | P3911 | |
Potassium Hydroxide (KOH) | Fisher Scientific | P250-1 | CAUTION KOH is an eye/skin irritant as a solid and corrosive in solution. Handle in a chemical fume hood using gloves, a lab coat, and goggles |
Protease Inhibitors | ThermoFisher Scientific | 78430 | |
ProteinA/G-MNase | Epicypher | 15-1016 | pA/G-MNase |
ProteinA-MNase, purified from pK19pA-MN | Addgene | 86973 | |
Proteinase K | New England Biolabs | P8107S | |
Qubit 1X dsDNA HS Assay Kit | ThermoFisher Scientific | Q33230 | |
Qubit Assay tubes | ThermoFisher Scientific | Q32856 | |
Qubit Fluorometer | ThermoFisher Scientific | Q33238 | |
Quick Ligase with 2X Quick Ligase buffer | New England Biolabs | M2200S | |
RNase A | New England Biolabs | T3010 | |
Sodium Acetate (NaOAc) | ThermoFisher Scientific | BP333-500 | |
Sodium Chloride (NaCl) | Sigma Aldrich | S5150-1L | |
Sodium dodecyl sulfate (SDS) | ThermoFisher Scientific | BP166-500 | SDS is poisonous if inhaled; handle with care in well ventilated spaces using gloves, eye protection, and an N95-grade respirator when handling |
Sodium Hydroxide (NaOH) | Fisher Scientific | S318-1 | NaOH is an eye/skin irritant as a solid and corrosive in solution. Handle in a chemical fume hood using gloves, a lab coat, and goggles |
Spermidine | Sigma Aldrich | S2626 | |
Standard Inverted Light Microscope | Leica | 11526213 | |
Standard lab agarose gel materials | Varies | ||
Standard lab materials such as serological pipettes and pipette tips | Varies | ||
T4 DNA Ligase | New England Biolabs | M0202S | |
T4 DNA Polymerase | New England Biolabs | M0203S | |
T4 PNK | New England Biolabs | M0201S | |
Tabletop vortexer | Fisher Scientific | 2215414 | |
Taq DNA Polymerase | New England Biolabs | M0273S | |
Thermomixer | Eppendorf | 5384000020 | Alternatively, can use a waterbath |
Tris base | Fisher Scientific | BP152-5 | |
Triton X-100 | Sigma Aldrich | 9002-93-1 | Triton X-100 is hazardous; use a lab coat, gloves, and goggles when handling |
Trypsin | Fisher Scientific | MT25052 | |
Tube rotator | VWR | 10136084 | |
USER enzyme | New England Biolabs | M5505S |