title: GSM3019328: TSC_ChIPseq_cFos; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP was performed by cross-linking proteins to DNA using 1% formaldehyde solution in J1 mouse ES cells. Briefly, cells were cross-linked with 1% formaldehyde for 7 min at room temperature, and then formaldehyde was quenched by adding glycine (final 125 mM) for 5 min. After washing cells with PBS two times, fixed cell pellets were resuspended in ChIP dilution buffer and sonicated using a Bioruptor (Diagenode) with a setting of 30 sec on and 1 min off for 10 min (3 times). Sheared chromatins containing average size of 300 bp DNA fragments were used for immunoprecipitation using 10ug of a native antibody against each factor. Immunoprecipitated DNA was sequenced using Illumina sequencers. Assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) was performed in in both ESCs and TSCs. Briefly, tagmentation reaction was performed with 50ng of target DNA, and then bPCR-compatible sites and barcode were added into the target DNA by PCR. After cleaning up the PCR products using Zymo DNA clean and concentrator-5 kit, the final product was sequenced using Illumina NextSeq 500 machine. Global gene expression profiles using RNA-seq were performed in both ESCs and TSCs.  Briefly, total RNAs were isolated using the RNeasy plus minikit (Qiagen). mRNAs were enriched from total RNAs, fragmented, and primed. First strand cDNAs were synthesized following by second strand synthesis. The ends of purified double strand cDNAs were repaired, ligated with a barcoded adaptor, and amplified with PCR. Purified final PCR product was sequenced using Illumina NextSeq 500 machine. ChIP-seq library  was constructed using the NEB ChIP-seq library prep. kit (E6200L ) following by manufacturer's instructions. ATAC-seq library was constructed using Nextera DNA library prep kit following the manufacturer's protocol. RNA-seq library was constructed using NEBNext Ultra RNA Library prep kit (NEB, E7530S) according to manufacturer's instruction ChIP-Seq, ATAC-seq, and RNA-seq
attributes.tag: GEO Accession
attributes.value: GSM3019328