title: GSM2829047: NANOG Hi-ChIP Rep1; Homo sapiens; OTHER
library_construction_protocol: For Hi-C, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched with glycine, nuclei were isolated and chromatin digested by HindIII, filled in with biotin-dCTP, and ligated. After ligation, nuclei were digested by ProteinaseK and chromatin were reverse-crosslinked overnight, followed by ethanol precipitation to get purified DNA. DNA were sonicated to 200-500 bp fragment and enriched with streptavidin dynal beads, followed by illumina library preparation. For ChIP-seq, nuclear lysates were sonicated to generate 200-500 bp fragments . Chromatin was precleared with Protein A or G Dynabeads at 4°C for 2 hours and incubated with antibody overnight at 4°C. Immunoprecipitated chromatin was eluted, reverse crosslinked and purified by standard methods, followed by illumina library preparation. For HiChIP nuclei were isolated and chromatin digested by DpnII, filled in with biotin-dCTP, and ligated. After ligation, chromatin was sonicated and precleared with Protein A and G Dynabeads at 4°C for 2 hours, then precipitated using Protein A or G dynalbeads pre-incubated with proper antibodies  overnight. Immunoprecipitated chromatin was eluted, reverse crosslinked and purified by standard methods, followed by streptavidin beads pull down and illumina library preparation. For ATAC-seq, H9 cells grown to exponential stage in 96-well plates cultured at 37°C or heat stressed at 42°C were collected and used for Fast-ATAC and OMNI-ATAC protocol. For fast-ATAC, cells on 96-well plate were washed with PBS and incubated in 50 µl transposes Tn5 mixture (0.01% digitonin for permeabilizing cell membrane, 2.5 µl Tn5, 25 µl TD buffer) at 37°C for 20 min with occasional shaking. For OMNI-ATAC, cells on 96-well plate were washed with PBS and nuclear membrane was disrupted by soaking in lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin) on ice, nuclei was washed once in cold lysis buffer without NP40 or digitonin, and then incubated in Tn5 transposes mixture (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O) at 37°C for 20 min with occasional shaking. After the reaction is over, DNA was directly extracted by Minelute Kit (Qiagen). Purified tagmented DNA were used to make illumina Nextera libraries. For eU-Seq, Ctrl and HS cells were labeled with EU in growth medium for 30 min at 37°C and 42°C respectively before RNA extraction with TRIZOL reagent. Purified RNAs were converted by click-chemistry into Biotin-U labeled RNAs and pulled down with streptavidin beads, followed by fragmentation and reverse transcription into cDNA. Illumina libraries were made from dsDNAs after second strand synthesis on cDNA and amplified with barcoded primers for sequencing. Libraries were constructed using the standard protocol. Genomic fragments were end repaired (NEBNext End Repair Module), A-tailed by adding  adenosine to  the 3' ends of fragment using Klenow fragment (3' to 5' exo minus, New England Biolabs), and adaptors were ligatedat room temperature for 1 hr with T4 DNA ligase (New England Biolabs). Libraries were amplified with Illumina primers using the KAPA SYBR FAST qPCR Master Mix. Nextera libraries were amplified with Nextera barcoded primers using the KAPA SYBR FAST qPCR Master Mix.
identifiers.namespace: GEO
identifiers.id: GSM2829047