alias: GSM3532108
title: GSM3532108: FX_FMRP_ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were thawed on ice with 1 ml Farham's lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, Halt protease inhibitor cocktail (Thermo Scientific)) for 5 m.  Nuclei were prepared by centrifuging the lysate at 2000 rpm for 5 m.  The nuclear pellet was resuspended in 300 μl RIPA (1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, Halt protease inhibitor cocktail).  Samples were sonicated in a Bioruptor (Diagenode) at high setting for a total time of 40 m with 30 s ON and 30 s OFF at 4°C.  The sonicated mixture was centrifuged at 13,500 rpm for 15 m at 4°C and the volume of supernatant was adjusted to 1 ml with RIPA buffer.  100 μl of this mixture was set aside as "input" and the remaining nuclear fraction was used for immunoprecipitation.  Both aliquots were snap frozen in liquid nitrogen and stored at -80°C until ready for immunoprecipation.  10 μg of monoclonal anti-FMRP antibody (Covance, now Biolegend, Cat#MMS-5231) was conjugate with M280 Dynabeads sheep anti-mouse IgG (Life Technologies).  The thriced washed beads (with PBS/BSA) were incubated with 900 μl sonicated nuclear fraction overnight at 4°C with agitation.  The beads were then washed 5 times with cold LiCl wash buffer (100 mM Tris pH7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate), followed by a single wash in TE (10 mM Tris-HCl pH7.5, 0.1 mM EDTA). The immunoprecipitates were eluted with 200 μl of IP elution buffer (1% SDS, 0.1 M NaHCO3) at 65°C for 2 h with vortexing every 30 m.  The 100 μl "input" was thawed and together with the eluted immunoprecipitates were reverse-crosslinked at 65°C overnight.  The samples were then used for ChIP-seq library construction. DNA was purified from all samples (input and immunoprecipitates) using Qiagen PCR clean-up kit and then end-repaired in a 100 μl reaction (1x End-repair buffer, 250 μM dNTP mix, 1 mM ATP, and 3 μl End-It Enzyme (Epicentre)) for 45 m at room temperature.  Labeled DNA was purified using Qiagen PCR clean-up kit, A-tailed using Klenow(exo-) (NEB E6055A), purified again with Qiagen PCR clean-up kit, and ligated to Ilumina adaptors using standard conditions.  Ligated DNA was purified with Qiagen PCR clean-up kit and amplified by PCR (18 cycles) using index primers (98°C, 5m; 18 cycles of 98°C, 20s; 65°C, 15s; 72°C, 1m; followed by 1 cycle of 72°C, 5m).  The PCR products were purified by AmPure beads (AgenCourt) to rmove free adaptors. 10E6 PBS-washed cells were embedded in 0.5% Incert low-melting point agarose in PBS and cast into plugs (0.5cmx1cmx1.5mm).  Plugs were then incubated in  6 ml lysis buffer (0.5M EDTA, 1% Sarkosyl, 200ug/ml Proteinase K) at 50 degree celcius for 48 h, with a buffer exchange at 24 h.  Plugs were then washed three times, for 1 h each, with 6 ml TE at room temperature. The DNA in TE-washed agarose plugs was then in-gel end-labeled with biotinylated dNTP mix (1 mM dTTP, dCTP, dGTP, 0.84 mM dATP, 0.16 mM Biotin-14-dATP) using End-It Kit (Epicentre). Plugs were then treated with beta-Agarase (NEB) to digest agarose and release DNA. DNA sample was then sonicated using a Covaris M220 using the snap-cap DNA 300bp shearing protocol.  DNA was then processed using a PCR Cleanup Kit (Qiagen) and run on agarose gel to verify the fragmentation pattern of DNA and quantified on a Nanodrop. 10-11 microgram of DNA was then end-repaired using a regular End-It kit and purified by the PCR Clean-up Kit.  The DNA was then A-tailed by A-tail Kit or exo-Klenow (NEB) and purified by PCR Clean-up kit, followed by quantification on a Nanodrop.  M270 Dynabeads (Life Technologies) were used to purify biotinylated DNA.  DNA bound to beads was calculated by measuring the quantity of DNA in the flow through.  DNA-bound beads were then resuspended in a T4-ligase mix containing Illumina adaptors.  400 ng of DNA bound to beads was used for PCR amplification using KAPA Hotstart Ready Mix (KAPA).  Each sample was given a specific index primer for multiplexing.  PCR product was then run on agarose gel to verify amplification and quantity.  AMPure beads (Agencourt) were used to remove free adaptors and the final product was analyzed on agarose gel. Break-seq libraries were sequenced on Illumina HiSeq 2500 with 100 or 150 bp paired-end reads, followed by Break-seq data analysis.
identifiers.namespace: GEO
identifiers.id: GSM3532108