GSM3712985: Input ChIPSeq 2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
HeLa-S3 cells
cell type
Human cervix epithelial cells
passenage
8
antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-seq: Chromatin preparation was essentially performed as previously described 36 with some modifications. Briefly, U937 and HeLa cells were protein cross-linked using 2 mM DSG for 45 min at RT. Cells were then washed twice with PBS, and DNA cross-linking was performed with 1% formaldehyde for 15 min (U937) or 10 min (HeLa) at RT with gentle shaking. Cross-linking reaction was quenched using 1.25 M glycine. Cells were then washed with PBS twice and harvested in Buffer B (20 mM HEPES, 0.25% Triton X-100, 10 mM EDTA, and 0.5 mM EGTA). Cells were pelleted by centrifuge at 2000 RPM for 5 min at 4 °C and resuspended in Buffer C (150 mM NaCl, 50 mM HEPES, 1 mM EDTA, and 0.5 mM EGTA). After that, cells were pelleted and resuspended in 1x incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 1mM EDTA, 0.5mM EGTA, and 20mM HEPES) at 15 million cells/mL. Cells were sheared in a Bioruptor Pico sonicator (Diagenode) at 4 °C using 6 (U937) or 7 cycles (HeLa) of 30 sec ON, 30 sec OFF. Sonicated material was centrifuged at 13000 RPM for 15 min at 4 °C, then stored at 80°C. RNA-seq: The total RNA of cells was extracted using Trizol (Life Technologies) according to manufacturer's instructions. After DNase treatment, 5 μg of extracted RNA was depleted from ribosomal RNA using Ribo-Zero Gold Kit (Epicenter Madison, Winsconsin, USA). After fragmentation of the rRNA-depleted RNA, 500ng was reverse-transcribed using Super Script III Reverse Transcriptase (Invitrogen) and random primers (Invitrogen) following the manufacturer's instructions. ChIP-seq: Ten million cells were used as input for library preparation, and 5x106 cells were used as input for ChIP-qPCR experiments. Chromatin was pre-cleaned by incubating with Protein A/G Dynabeads (Invitrogen) for 90 min at 4 °C in 1x incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES) supplemented with protease inhibitors and 0.1% BSA. Chromatin was then incubated with 5 μg of antibody (anti-BRD9, Active Motifs, catalog number: 61538) overnight at 4 °C in 1x incubation buffer supplemented with protease inhibitors and 0.1% BSA. Protein A/G Dynabeads were added the day after followed by a 90 min incubation. The beads were washed twice with Wash Buffer 1 (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), once with Wash Buffer 2 (Wash Buffer 1 with 500 mM NaCl), once with Wash Buffer 3 (250 mM LiCl, 0.5% sodium deoxycholate, 0.5% NP-50, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), and twice with Wash Buffer 4 (1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES). After washing, beads were rotated for 30 min at RT in Elution Buffer (1% SDS, 0.1 M NaHCO3). The supernatant was decross-linked with 200 mM NaCl and 100 μg/mL Proteinase K over night at 65 °C. Decross-linked DNA was purified by MinElute PCR Purification columns (Qiagen). DNA amount was quantified using Qubit fluorometric quantitation (Thermo Fisher Scientific). ChIP-seq libraries were prepared using the Kapa Hyper Prep Kit for Illumina sequencing (Kapa Biosystems) according to the manufacturer's protocol with the following modifications. 5 ng ChIP DNA was used as input, with NEXTflex adapters (Bioo Scientific) and 10 cycles of PCR amplification. Post-amplification clean-up was performed with QIAquick MinElute columns (Qiagen) and size selection was done with an E-gel (300 bp fragments) (Thermo Fisher Scientific). Size-selected samples were analyzed for purity using a High Sensitivity DNA Chip on a Bioanalyzer 2100 system (Agilent). Samples were sequenced on an Illumina HiSeq2000 or NextSeq500. The 43 or 75 bp tags were mapped to the reference mouse genome mm9 (NCBI build 37) or Drosophila genome dm3 (for spike-in) using the Burrows-Wheeler Alignment tool (BWA) allowing one mismatch38. Only uniquely mapped reads were used for data analysis and visualization. Mapped reads were filtered for quality and duplicates were removed. Peak-calling was performed with the MACS 2.0 tool against a reference input sample from the same cell line39. RNA-seq libraries were prepared using the TruSeq RNA Sample Prep Kit (Illumina) following the manufacturer's instructions. Libraries were indexed using NEXTflex adapters (Bioo- Scientific Corporation, Austin, TX, USA), and the quality was assessed by qPCR and Bioanalyzer (BioRad). Single-end 43bp deep sequencing was performed on Illumina instruments using TruSeq reagents (Illumina, San Diego, CA, USA), according to manufacturer's instructions.