Di-Methyl-Histone H3 (Lys27) (D18C8) XP® Rabbit mAb #9728

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.	-For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. -It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.	-Once in solution, store 1M DTT at -20°C.

Upstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.
Protocol tips
-For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. -It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.
Downstream tips
-Once in solution, store 1M DTT at -20°C.

Publication protocol

Cells (cell lines or dissociated tumor cells) were fixed with 1% formaldehyde (Sigma). Fixed cell preparations were washed, pelleted and stored at −80 °C. Sonication of lysed nuclei (lysed in a buffer containing 1% SDS) was performed on a BioRuptor UCD-300 for 60 cycles, 10 s on 20 s off, centrifuged every 15 cycles, chilled by 4 °C water cooler. Samples were checked for sonication efficiency using the criteria of 150–500 bp by gel electrophoresis. After the sonication, the chromatin was diluted to reduce SDS level to 0.1% and before ChIP reaction 2% of sonicated drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing (see below).

ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit. Twenty-five microliter Protein A beads (Invitrogen) were washed and then incubated with antibodies (anti-H3K27M (1:66, Millipore ABE419), anti-H3K27me3 (1:40, CST 9733), anti-H3K27me3 (1:100, Active Motif 61017), anti-H3K27me2 (1:50, CST 9728), anti-H3.3 (1:66, Millipore 09–838), anti-HA(1:100, CST 3724)) as listed in Supplementary Table 6, and 2 million cells of sonicated cell lysate combined with protease inhibitors for 10 h, followed by 20 min wash cycle with provided wash buffers.

ChIP reaction for SUZ12 and RING1B was performed as follows: antibodies (anti-SUZ12 (1:150, CST 3737), anti-RING1B (1:200, Active Motif 39663)), also listed in Supplementary Table 6) were conjugated by incubating with 40 ul protein A or G beads at 4 °C for 6 h, then chromatin from ~4 million cells was added in RIPA buffer, incubated at 4 °C o/n, washed using buffers from Ideal ChIP-seq Kit (one wash with each buffer, corresponding to RIPA, RIPA + 500 mM NaCl, LiCl, TE), eluted from beads by incubating with Elution buffer for 30 min at room temperature.

Reverse cross linking took place on a heat block at 65 °C for 4 h. ChIP samples were then treated with 2 ul RNase Cocktail at 65 °C for 30 min followed by 2 ul Proteinase K at 65 °C for 30 min. Samples were then purified with QIAGEN MiniElute PCR purification kit as per manufacturers’ protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol.

Library preparation was carried out using Kapa HTP Illumina library preparation reagents. Briefly, 25 ul of ChIP sample was incubated with 45 ul end repair mix at 20 °C for 30 min followed by Ampure XP bead purification. A tailing: bead bound sample was incubated with 50 ul buffer enzyme mix for 30 °C 30 min, followed by PEG/NaCl purification. Adaptor ligation: bead bound sample was incubated with 45 ul buffer enzyme mix and 5 ul of different TruSeq DNA adapters (Illumina) for each sample, for 20 °C 15 min, followed by PEG/NaCl purification (twice). Library enrichment: 12 cycles of PCR amplification. Size selection was performed after PCR using a 0.6 × /0.8x ratio of Ampure XP beads (double size selection) set to collect 250–450 bp fragments.

ChIP libraries were sequenced using Illumina HiSeq 2000, 2500, or 4000 at 50 bp single reads.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Di-Methyl-Histone H3 (Lys27) (D18C8) XP® Rabbit mAb #9728 below.

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