GSM1325073: input replicate2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Lung
Cell type
IMR-90
Primary Tissue
Lung
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
source_name
IMR90_input
cell line
IMR90
cell type
human primary lung fibroblasts
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were grown to 80 to 90% confluence in 15cm dishes, cross-linked with 1% formaldehyde in PBS at room temperature for 10 min, then quenched in 125 mM glycine in PBS at room temperature for 5 min. The cells were washed by cold PBS once and were collected by centrifugation and sonication in lysis buffer (50mM Tris pH 7.9, 10 mM EDTA, 1% SDS, 1x protease inhibitor cocktail and 1mM DTT) to generate chromatin fragments of ~500 bp in length. The material was clarified by centrifugation 14,000rpm for 10min at 4 °C, and 20 µl supernatant was used as input for quantitation, the remaining supernatant was diluted 10-fold in dilution buffer (20 mM Tris pH 7.9, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 1X protease inhibitor cocktail and 1mM DTT), and pre-cleared by incubation with protein A-agarose beads at 4°C for 3hr. The pre-cleared, chromatin-containing supernatant was used in immunoprecipitation reactions at 4°C overnight with an antibody against macroH2A1, then incubated with protein A-agarose beads at 4°C for 2hr. No antibody negative controls were included for every ChIP experiment. The immuneprecipitated DNA was cleared of protein by digestion with 0.4mg/ml glycogen and proteinase K (2.5U/ml, Roche) in Txn stop buffer (20 mM EDTA, 0.2 M Nacl and 1% SDS) at 37°C for 1 hour. The DNA was then extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitated. DNA fragments from ChIP reactions were repaired using T4 DNA Polymerase (NEB) and T4 DNA Polynucleotide kinase (NEB) in T4 DNA ligase buffer (NEB). Next, dA ends were generated by employing Klenow Fragment (3’->5’ exo-, NEB), in Buffer 2 (NEB). Adapter ligation reactions were then set up using indexed adapters and Quick Ligase (NEB). Ligation-mediated PCR was then performed using Phusion High-Fidelity DNA Polymerase (NEB). The libraries were visualized on 2% E-Gel® General Purpose Agarose Gels (Invitrogen). Sequencing for the macroH2A1 ChIPs were performed on an Illumina HiSeq 2500 at the Einstein Epigenomics Core Facility. Sequences reads were mapped to the human genome version hg19 using ELAND version 1.7.0 (Illumina).