1. ULI-NChIP PROCEDURE
A. SAMPLE ISOLATION BY FLOW CYTOMETRY
As ULI-NChIP is dilution-based, the conditions of cell isolation and storage are important for the subsequent steps in the procedure. When the expected isolated cell number is sufficient for the formation of a visible cell pellet, we suggest sorting the cells directly in PBS containing protease inhibitor cocktail and pelleted prior to flash freezing and storage. However, sample loss is much higher when the isolated cell number is insufficient for the formation of a visible pellet and thus, rarer cell types can be sorted and stored directly in the nuclear isolation buffer that will be used later during micrococcal nuclease (MNase) digestion of chromatin (B). There is a certain degree of flexibility in the isolation condition, and cell numbers higher than 2 × 104 cells can also be sorted directly in nuclear isolation buffer, as long as the volume of nuclear isolation buffer is appropriately adjusted.
When expecting > 2 × 104 cells
- Label clean 1.5 ml sample tubes.
- Add 100 μl of PBS containing 1x protease inhibitor cocktail.
- Sort desired cell population directly in the 1.5 ml sample tubes.
- Pellet cells by centrifugation (500 g for 5 minutes at 4°C).
- Remove supernatant.
- Flash freeze cell pellets by immersing sample tubes in liquid nitrogen for 0.5 minute.
- Store samples at -80°C. Cell pellets can be stored from a few weeks to a few years.
When expecting < 2 × 104 cells
- Label clean 1.5 ml sample tubes.
- Add 10-20 μl of complete nuclear isolation buffer to each tube. Suggested volumes of buffer per expected sample size are presented in Table 1.
- Sort desired cell population directly in complete nuclear isolation buffer.
- Estimate the total volume of sample, cells and sheath buffer. The contribution of sheath buffer to the final volume should remain below 1/3 of the final volume.
- [optional] If sheath buffer contribution is above 1/3 of the final volume:
i) Pellet cells by centrifugation (500 g for 5 minutes at 4°C).
ii) Remove all but 10 μl of nuclear isolation buffer + sheath buffer.
iii) Add 10 μl of complete nuclear isolation buffer.
- Flash freeze samples in complete nuclear isolation buffer by immersing sample tubes in liquid nitrogen for 0.5 minute.
- Store samples at -80°C. Samples can be stored from a few weeks to a few years, and be pooled together in subsequent steps.
Table 1: Suggested conditions for cell isolation and storageSee figure in Figures section.
B. CHROMATIN PREPARATION
Preparation of MNase fragmented chromatin is one of the most critical steps of native ChIP. Insufficiently digested chromatin will lead to input bias and higher background levels, while the yield of overdigested chromatin will be reduced.
A small amount of detergents is added to the nuclear isolation buffer to increase accessibility of MNase to the chromatin, and PEG 6000 is included as a crowding agent to allow for more uniform digestion of smaller inputs. Please note that MNase is very sensitive to temperature changes, and will lose efficiency with time. Small aliquots of MNase should be prepared and transported to the bench no more than 2-3 times for consistent results. It is recommended to test each new batch of MNase with low numbers of cultured cells prior to use for in vivo samples.
Thaw frozen cell pellet or, for lower cell numbers, cells in nuclear isolation buffer. At this stage, samples isolated on different days can be pooled together.
[optional] If cells are frozen as a pellet, re-suspend in appropriate volume of complete nuclear isolation buffer ( Table 2 ).
Pipette samples up and down 15-20 times while swirling and place back on ice.
Prepare dilute MNAse stock enzyme in MNAse dilution buffer to 200 U/μl (0.5 µl stock in 4.5 µl MNase dilution buffer).
!CAUTION! Add MNase enzyme only when you are ready to proceed to digestion.
Prepare MNAse digestion buffer as described in Table 2 (always prepare just before digestion).
Place samples on rack at 21-25°C.
Add MNase master mix (+MNase enzyme) to each sample. Mix very well (15-20 times) with pipettor.
!CAUTION! When digesting multiple samples at the same time, it is important to leave the MNase master mix on ice.
Allow reaction to proceed according to the conditions suggested in table 2 .
Stop reaction by adding 10% of the reaction volume of 100 uM EDTA and mix very well with pipette (20-30 times). Place samples immediately on ice.
Add 1% Triton/1% deoxycholate solution as per indicated in Table 2 .
Rest samples on ice for 15 minutes.
Vortex samples (medium setting) for approximately 30 seconds.
Add complete immunoprecipitation buffer to the digested chromatin. Digested chromatin should take < 25% of the immunoprecipitation volume. Depending on the amount of cells available and digestion volume, immunoprecipitation will be performed in a 100-200 μl final volume.
Rotate chromatin at 4°C for 1 hour.
Vortex (medium setting) for 30 seconds
Take out an input control aliquot (for small samples, inputs tend to be larger than for normal ChIP, to reduce errors due to low cell numbers. For < 20,000 cells input per immunoprecipitation, the input should be approximately 10% of the sample).
This input can be readily extracted if you want to control for fragmentation size. Add 10% volume of 10% SDS, mix well and add EB to obtain a final volume of 100 μl. Proceed to DNA extraction as described in Section D.
Table 2: Suggested conditions for MNase digestion of native chromatinSee figure in Figures section.
C. CHROMATIN IMMUNOPRECIPITATION
A challenge of performing ChIP on very small samples is finding the balance between obtaining sufficient material for sequencing and using conditions that are stringent enough to reduce the unavoidable background associated to such samples. A good knowledge of the antigen being pulled down and of the antibody used should guide the experimental design. Antigen that can be pulled down at a higher frequency allow for the use of smaller samples, while it may be difficult to generate high resolution enrichment profiles of a mark which is present in only a small percentage of a cell population. This protocol has been successfully used down to 103 cells using the following antibodies; H3K9me3 (Active Motif #39161), H3K27me3 (Diagenode pAb-069-050), H3K36me3 (Abcam #9050) and H3K4me1 (Upstate #07-436). This protocol has been successfully used down to 5 × 103 cells using the following antibody; H3K4me3 (Abcam #1012), but remains to be tested for smaller inputs.
- Pre-wash Protein A/G magnetic beads (Dynabeads, Life Technologies #1006D and 1007D) 3 times in complete immunoprecipitation buffer. Each immunoprecipitation will require 5 μl (<10,000 cells input) or 10 μl (10-100,000 cells input) of beads for pre-clearing the chromatin and 5-10μl of beads for immunoprecipitation.
- Prepare antibody beads complexes (as described in Table 3 (A) ) in 200 μl PCR strip tubes. Rotate at 4°C for 3-8 hours.
- Pre-clear chromatin ( Table 3 (B) ) prepared in section B: add 5 or 10µl of pre-washed magnetic beads per immunoprecipitation, depending on input.
- Rotate for 2-6 hours at 4°C.
- After incubation, place antibody-beads complexes ( Table 3 (A) ) on magnetic rack and take out supernatant.
- Place pre-cleared chromatin ( Table 3 (B) ) on a magnetic rack. Transfer chromatin (supernatant) to antibody-beads complexes.
- Rotate overnight at 4°C.
- Place PCR strip tubes on a magnetic rack.
- Discard unbound chromatin.
- Re-suspend the beads in complete immunoprecipitation buffer and transfer to labeled 1.5 ml sample tubes.
- Place on a magnetic rack. Remove supernatant.
- Wash the antibody-beads complexes as follows:
i) Re-suspend beads in 200 μl low salt wash buffer. Take out the wash buffer and repeat.
ii) Re-suspend beads in 200 μl high salt wash buffer. Take out the wash buffer.
iii) Re-suspend beads in 200 μl high salt wash buffer. Transfer beads to a clean 1.5 ml sample tube.
iv) Take out wash buffer. Close the tubes and pulse-spin the beads. Use a gel loading pipette tip to remove the last few drops of wash buffer.
- Re-suspend the antibody-beads complexes in 30 µl freshly prepared ChIP elution buffer.
- Elute DNA for 1-1.5 hours in a 65°C water bath. Vortex tubes regularly.
- Pulse-spin the sample tubes and place on a magnetic rack.
- Transfer the eluted chromatin to a clean 1.5 ml sample tube.
- Wash the beads with 70 µl of EB and combine with the eluted chromatin.
Table 3: Suggested conditions for native ChIPSee figure in Figures section.
D. DNA EXTRACTION
Various methods can be used for extraction and purification of DNA from chromatin samples. As a general rule, it is preferable to avoid any DNA purification procedure that is column-based, as a significant proportion of smaller samples will not be recovered.
Transfer eluted chromatin to a pre-spun phase lock tube.
Add 100 µl of phenol: chloroform: isoamyl alcohol 25:24:1 to each sample. Vortex vigorously for 20-30 seconds.
Spin at 13,000g for 5 minutes at 21-25°C.
Transfer the upper phase to a new 1.5 ml tube.
!CAUTION! Make sure to take all the liquid and to not touch the new tube with the tip of the pipette (which could transfer some of the grease in the following reaction).
Add 10 µl of 3M sodium acetate and 1 µl of LPA (linear polyacrylamide). Mix well. (LPA can be replaced by glycogen as a co-precipitating agent).
Add 275 µl of cold Ultrapure Ethanol (keep at -20°C).
Mix very well and allow DNA to precipitate for at least 30 minutes (can be overnight) at -20°C.
Spin down DNA at 13,000g for 30 minutes at 4 degrees.
Take out supernatant and wash the pellet with 200 µl of freshly prepared 70% ethanol.
Allow pellet to dry.
Re-suspend in 20 (input < 20,000 cells) or 30 (input > 20,000 cells) μl of buffer EB.
NOTE At this point, if the DNA is re-suspended in a low volume, the concentration of salts/SDS present will interfere with qPCR or the first step of library construction. We recommend re-purifying the DNA with 1.8 volumes of Agencourt Ampure XP beads.
NOTE RNAse A treatment is recommended prior to qPCR, depending on amplicon. For 1,000 cells ChIP, we typically take out 10-15% of the eluate and dilute it for qPCR. We can run 2-3 multi-copy amplicons with such an aliquot, but there is typically not enough material for single copy genes.
E. DNA PURIFICATION USING AMPURE XP BEADS
- Make sure the beads are well re-suspended. Add 1.8x volume (36 μl if sample was re-suspended in 20 μl EB) of bead slurry per sample.
- Mix very well with a pipette (10-12 times). Once the beads have been added to all samples pulse-vortex 3-4 times.
- Incubate at room temperature for 10 minutes.
- Place the samples on a magnetic rack for 2 minutes or until the sample is clear of beads.
- Remove the liquid from the beads.
- Wash the beads (leave on the magnetic rack) twice with 100 µl of freshly prepared 70% ethanol.
- Take out the ethanol, pulse-spin and remove the last bit of ethanol using a gel loading tip.
- Allow the beads to air dry.
- Add 20 (< 20,000 cells input) or 30 (>20,000 cells input) μl of buffer EB to the beads, making sure they are well covered.
- Allow beads to rehydrate at room temperature for approximately 5 minutes (they are rehydrated when you can re-suspend them by flicking the tube). Pipette up and down 15-20 times to re-suspend completely.
- Place the tubes back on the magnetic rack.
- Transfer the eluted DNA buffer to a new tube, taking care not to transfer any magnetic beads.
2. LIBRARY CONSTRUCTION
Raw ChIP material generated by ULI-NChIP-seq will range from a few pg to a few ng of DNA, depending on the input size and antigen. There are various methods and kits that can be used for construction of libraries from low inputs of material. Described here is an adapter ligation-based followed by PCR amplification library construction procedure that works from picograms of DNA material. Indexed libraries can be pooled prior to size selection, thus reducing sequencing costs and the number of PCR cycles required to generate sufficient amounts of material for sequencing.
F. END REPAIR OF FRAGMENTED DNA
- Thaw ChIP samples (in 20 or 30 µl of EB buffer). Keep on ice until End Repair Master Mix is prepared.
- Prepare the End Repair Master Mix in a clean 1.5 ml tube as follows:
See figure in Figures section.
NOTE 10X Phosphorylation buffer is T4 DNA ligase buffer (NEB B0202S)
- Mix thoroughly and pulse spin. Keep master mix on ice while distributing to samples.
- Add 5 (if <10,000 cells per ChIP) or 7.5 µl (if >10,000 cells per ChIP) of End Repair Master Mix to each sample tube. Mix very well (10-15 times) using pipettor.
- Incubate at room temperature for 30 minutes.
- Proceed to DNA purification using Ampure XP beads (steps 52-63). In step 60, elute DNA in 17 (< 20,000 cells input) or 25.5 (> 20,000 cells input) µl of EB.
Set the heating block or waterbath at 37°C.
Thaw and pulse-spin end-repaired (or digested with blunt cutter) samples (in 17 or 25.5 µl buffer EB).
Prepare A-tailing Master Mix in a clean tube as follows:
See figure in Figures section.
Vortex and pulse-spin A-tailing Master Mix and add 3 or 4.5 µl to each sample. Mix thoroughly by pipetting/stirring.
Incubate at 37°C for 30 minutes.
Proceed to DNA purification using Ampure XP beads (steps 52-63). In step 60, elute DNA in 6.67 (< 20,000 cells input) or 10 (> 20,000 cells input) µl of EB.
H. ADAPTER LIGATION
Prepare Adaptor Ligation Master Mix in a clean tube as follows:
See figure in Figures section.
Add 13.34 or 20 μl of Adaptor Ligation Master Mix to each sample, mixing very well.
Incubate at 21-25°C for at least 30 minutes. Can be extended to O/N ligation.
Proceed to DNA purification using Ampure XP beads (steps 52-63). In step 52, add 0.8x volume (16 or 24 µl of Ampure XP beads). In step 60, elute DNA in 11 µl of EB.
I. LIBRARY AMPLIFICATION
Prepare PCR Master Mix as follows (keep on ice until use):
See figure in Figures section.
Distribute 1 µl of the appropriate indexed primers PE 2.x into a PCR plate or 8-tubes strips.
Distribute 13.5 µl of PCR Master Mix into sample tubes.
Add 10.5 µl of adaptor-ligated samples to PCR tubes and mix very well (10-20 times) with pipette.
Pulse-spin tubes or plate
Run following PCR program (select appropriate, depending on the material to amplify, suggested in Table 8):
× 10-18 amplification cycles (see Table 8)
Table 8: Suggested number amplification cycles of constructed libraries depending on input size and immunoprecipitated chromatin markSee figure in Figures section.
- Proceed to DNA purification using Ampure XP beads (steps 52-63). In step 52, add 0.8x volume (16 or 24 µl of Ampure XP beads). In step 60, elute DNA in 15 µl of EB.
- Evaluate library yield and quality using an Agilent Bioalalyzer (High Sensitivity DNA detection kit) or Agilent TapeStation (D1000 High Sensitivity screentape).
NOTE Due to the small amount of template in the library construction procedure, it is normal at this stage to observe a large amount of adapter dimers. These will be removed prior to sequencing.
3. LIBRARY POOLING AND SIZE SELECTION
Libraries should be pooled according to the number of reads that are required. As low input libraries will have a tendency to have a higher proportion of unmapped and duplicate reads, it is a good idea to sequence deeper than the number of distinct reads required. As low-input samples are often from precious samples, we recommend using paired-end sequencing, which will enable inferring exact fragments size as reducing the number of reads that will be flagged as duplicates. For heterochromatic marks such as H3K9me3 and H3K27me3, we recommend sequencing 30-40 million read pairs (60-80 million reads). For H3K4me3, sequencing 5-15 million read pairs (10-30 million reads) should yield sufficient distinct reads to generate high resolution peaks.
- Pool libraries to be sequenced together according to their molarity and the desired number of reads.
- Run the pooled libraries on an agarose gel. In order to prevent having constructed library fragments floating around and potentially contaminating experiments, we recommend using a buffer-free system such as E-gel (Life Technologies).
- Cut out the 250-650 bp fragments from the gel and transfer to a new sample tube.
- Proceed to gel extraction of DNA and elute in 15 µl of EB. We recommend the Zymoclean Gel DNA Recovery kit from Zymo Research.
- Confirm the absence of adapter dimers (~ 130 bp) in the purified library pool using an Agilent Bioalalyzer (High Sensitivity DNA detection kit) or Agilent TapeStation (D1000 High Sensitivity screentape).