title: GSM6102702: H3K4me3 4; Homo sapiens; ChIP-Seq
library_construction_protocol: Eight mL fresh blood was collected from FXS male (N=10) and age-matched typically developing males (N=7) individuals (See Supplemental Table 1) in a BD vacutainer CPT (Cell Preparation Tube with sodium citrate- blue top tube, Becton Dickinson #REF362761). The tube was gently inverted 5 times and the sample was centrifuged for 25 minutes at 1500-1800 RCF at room temperature. The tubes were then inverted to collect the lymphocytes and other mononuclear cells resuspended in the upper liquid phase in a new 15mL tube. The samples were centrifuged again for 10 minutes at 300 RCF to obtain the PBMC (peripheral blood mononuclear cells) pellet. The PBMCs were rinsed with 1X Dublecco's phosphate buffered saline w/o calcium or magnesium (D-PBS) (Invitrogen #14190-094). The PBMC pellet was resuspended in 250uL ice-cold D-PBS with protease inhibitors. Chromatin isolation and sequencing was performed as previously described (3). Briefly the cells were cross-linked with 1% formaldehyde and quenched with 150mM glycine. After centrifugation at 2000g for 10 min at 40C the cells were lysed. After homogenization the nuclei were harvested by centrifugation at 2000g for 5min at 40C. The nuclei were lysed by incubating for 20 mins on ice in nuclear lysis buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 mM EGTA). 0.5% SDS was added and the samples sonicated on a Bioruptor® sonicator at high power settings for 9 cycles (sonication: 30 sec on, 90 sec off) of 15min each at 4°C. The samples were centrifuged and diluted to adjust the SDS concentration to <0.1%. 10% of each sample was used as input. The remainder of the samples were divided into two and incubated with protein G dynabeads coupled overnight at 40C with antibodies against H3K36me3 (Abcam ab9050, 5μg per ChIP) or H3K4me3 (Active Motif- 39159, 5 μg per ChIP). After IP, the beads were washed and chromatin and de-crosslinked overnight at 65°C. After RNAse and proteinase K treatment the DNA was purified. ChIP-Seq libraries were prepared by performing the following steps: ends repair using T4 DNA polymerase, A' base addition by Klenow polymerase and Illumina adapter ligation using T4 Polynucleotide kinase from New England Biolabs (NEB). The library was PCR amplified using multiplexing barcoded primers. The libraries were pooled with equal molar ratios, denatured, diluted, and sequenced with NextSeq 500/550 High Output Kit v2.5 (Illumina, 75bp paired-end runs,) on a Nextseq500 sequencer (Illumina).