title: GSM3145590: C4-2 [RNA-seq]; Homo sapiens; RNA-Seq
library_construction_protocol: For RNA-seq total RNA was extracted using the RNeasy Kit (Qiagen). For ChIP-seq 5-8 × 10^6 cells were fixed by adding 1:20 volume of fixing buffer (11% paraformaldehyde (Electron Microscopy Sciences, cat# 15714), 0.1M NaCl, 1mM EDTA pH 8.0, 50mM HEPES pH 8.0) directly to the tissue culture medium for 10 min at room temperature for histone modification ChIPs or at 37°C for transcription factor ChIPs and subsequently quenched by glycine. Cells were lysed in lysis buffer (50mM HEPES pH 8.0, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) for 10 min at 4°C. Nuclei were pelleted and washed in wash buffer (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) for 10 min at 4°C and then pelleted and resuspended shearing buffer (10mM Tris-HCl pH 7.4, 1mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 0.25% sarkosyl, 1mM DTT) and sonicated in a Covaris sonicator. Debris was removed by centrifugation (5 min at 10,000g) and 970µl of the lysate was mixed with 30µl of 5M NaCl. Chromatin was pre-cleared for 1 hr at 4°C with 40µl of Dynabeads Protein G (LifeTechnologies, cat# 10003D) washed in 0.5% BSA in PBS. Lysates were then incubated with corresponding primary antibody overnight at 4°C. Complexes were precipitated by incubation with 40µl of washed Dynabeads Protein G for 2 hrs at 4°C, washed with low salt wash buffer (20mM Tris-HCl pH 8.0, 150mM NaCl, 2mM EDTA pH 8.0, 1% Triton X-100, 0.1% SDS), high salt wash buffer (20mM Tris-HCl pH 8.0, 500mM NaCl, 2mM EDTA pH 8.0, 1% Triton X-100, 0.1% SDS), LiCl wash buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1mM EDTA pH 8.0, 1% NP40, 1% sodium deoxycholate), and twice with TE pH 8.0. DNA was eluted in elution buffer (100mM NaHCO3, 1% SDS) by incubating overnight at 65°C, followed by 30 min treatment with RNase A at 37°C and 2 hrs treatment with proteinase K at 55°C. DNA was purified with phenol-chloroform extraction and precipitated with isopropanol. For methylation analysis, DNA was isolated using QIAamp DNA Mini kit (QIAGEN, cat# 51304). DNA methylation profiling was carried out on Infinium HumanMethylation450K BeadChip arrays (Illumina, discontinued) at the Harvard Medical School-Partners HealthCare Center for Genetics and Genomics. RNA-seq libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kits from 500 ng of purified total RNA according to the manufacturer's protocol. The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent TapeStation 2200, and RT-qPCR using the Kapa Biosystems library quantification kit according to manufacturer's protocols. ChIP-seq libraries were prepared using the Rubicon ThruPLEX DNA-seq Kit from purified ChIP DNA or input DNA according to the manufacturer's protocol.
attributes.tag: GEO Accession
attributes.value: GSM3145590