title: GSM4387085: ATAC TKO_PP 2; Homo sapiens; ATAC-seq
library_construction_protocol: Total RNA was isolated using the Trizol reagent following the manufacturer's instructions. RNA integrity was determined using Agilent 2100 Bioanalyzer (Agilent Technologies). Subsequently, polyA tailed RNA were selected using the Dynabeads oligo(dT) (Thermo Fisher Scientific). Total gnomic DNA was isolated using the DNeasy Blood & Tissue kit (Qiagen). RNA libraries were prepared using the NEBNext® Ultra™ RNA Library Prep kit for Illumina (New England Biolabs). ATAC-seq libraries were prepared as previously described60. In brief, cells were enzymatically dissociated and lysed in lysis buffer (10 mM Tris-HCl, PH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Immediately after centrifugation, transposition reactions were carried out by adding Tn5 Transposes from the Illumina Nextera DNA library preparation kit to the isolated nuclei and incubated at 37 oC for 30 min. DNA fragments were purified using the MinElute PCR Purification kit (Qiagen) and amplified using the KAPA real-time library amplification kit. Libraries were purified using the PCRClean DXTM beads (Aline Biosciences).  CMS-IP-seq libraries were performed as previously described with small modifications (Huang et al., 2012). Purified genomic DNA (with 5% of mouse DNA and 0.5% lambda DNA spike-in) was sheared to 200–500 bp fragments using the M220 Focused-ultrasonicator (Covaris). Bisulfite converted DNA libraries with methylated adapters were enriched using an in-house anti-CMS antibody bound to protein A/G dynabeads. Amplified libraries were purified by AmpuXP beads (Beckman Coulter) and then sequenced using the Illumina NextSeq (75 and 40 cycle, single-end) systems. Genomic DNA was isolated using the DNeasy Blood & Tissue kit (Qiagen). WGBS library preparation and sequencing were conducted by BGI (Shengzhen, China). In brief, purified genomic DNA (with 1% of unmethylated lambda DNA spike-in, Promega) was sheared to a fragment size of 100–700 bp (main size 250bp) using Covaris focused ultrasonicator according to the manufacturer's instructions. DNA fragments were subjected to bisulfited conversion using the EZ DNA Methylation-gold kit (Zymo Research). Bisulfite converted DNA was amplified and finally subjected to sequence on a MGISEQ-2000 system (100-cycle, paired-end) at BGI (Shengzhen, China).  ChIP-seq was performed for TET1 (WT_PP cells), H3K4me1 (WT_PP and TKO_PP cells), and H3K27ac (WT_PP and TKO_PP cells). For TET1 ChIP-seq, approximately 5 x 107 cells were crosslinked, washed and snap frozen following the Cell Fixation protocol from Active Motif. TET1 ChIP-seq library preparation and sequencing were conducted by Active Motif. Chromatin immunoprecipitation of H3K4me1 and H3K27ac were performed as previously described28. WT_PP and TKO_PP cells were fixed, washed, and lysed in nuclear lysis buffer (50 mM Trish HCl, pH 8.0, 5mM EDTA, 1% SDS, and 1 protease inhibitor cocktail). Chromatin was sheared to 200-500 bp fragments using Bioruptor® (Diagenode), and the DNA fragments were precipitated with appropriate antibodies. ChIP-seq libraries were prepared using the NEBNext® Ultra IITM DNA Library Prep kit (New England Biolabs) following the manufacture's instruction and subjected to high throughput sequencing on a Illumina HiSeq 2500 system (150 bp, paired-end) at Novogene (Tianjin, China).
attributes.tag: GEO Accession
attributes.value: GSM4387085