title: GSM2760347: CLL-B01_RNA-Seq-Notch1WT-ICN1low; Homo sapiens; RNA-Seq
library_construction_protocol: ChIP-Seq: Cells were crosslinked by addition of 1% formaldehyde to media at 37 C for 10 minutes, followed by glycine quenching and 2x washing in PBS. Nuclei were isolated by hypotonic buffer lysis, and chromatin was fragmented with a Branson sonifier in a 1% SDS-containing lysis buffer. Samples were diluted 1:10 and chromatin complexes containing the target of interest were isolated by immunoprecipitation, followed by reverse crosslinking and DNA purification by SPRI. For input samples, 1:10 diluted sonicated chromatin (whole cell extract; wce), was added directly to the reverse crosslinking step without immunoprecipitation. RNA-Seq: Cells were pelleted and flash-frozen in liquid nitrogen. RNA was extracted from pellets via Qiagen Qiashredder and RNAeasy columns. ChIP-Seq: Purified ChIP or input DNA was end-repaired and A-tailed. Illumina barcoded adaptors were ligated and PCR amplified. Final library was isolated by double SPRI purification (sequential reverse and forward SPRI) and paired-end sequenced (38 bp x 2) on illumina NextSeq. RNA-Seq: Strand-specific RNA-Seq libraries were generated as follows: Oligo dT beads were used to isolate the poly-adenylated fraction of total RNA, followed by zinc-mediated fragmentation and DNAase treatment. A 3' single-stranded RNA adaptor was ligated to RNA fragments, and an adaptor-seated primer used for first-strand cDNA synthesis. RNA was then hydrolyzed, and a 3' DNA adaptor was ligated to ssDNA fragments, which were then PCR enriched with barcoded, adaptor-seated illumina PCR primers. Final libraries were isolated by SPRI purification (forward SPRI x2), and paired-end sequenced on illumina NextSeq (38 bp x2).
attributes.tag: GEO Accession
attributes.value: GSM2760347