title: GSM3455704: Elf FLCN KO #6 +AAVS1-FLCN-GFP + Dox 4 days 2iLif rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA for samples in this study were extracted with the Direct-zol RNA kit (Zymo Research) and libraries for Illumina sequencing were generated with the KAPA Stranded mRNA-Seq Kit (KAPA Biosystems). Sequencing was run on an Illumina HiSeq 2500 in the Fred Hutchinson Cancer Research Center Shared Resources Genomics facility. RNA libraries were prepared for sequencing using standard Illumina protocols Total RNA for samples in this study were extracted with the Direct-zol RNA kit (Zymo Research) and libraries for Illumina sequencing were generated with the KAPA Stranded mRNA-Seq Kit (KAPA Biosystems). Sequencing was run on an Illumina HiSeq 2500 in the Fred Hutchinson Cancer Research Center Shared Resources Genomics facility. RNA libraries were prepared for sequencing using standard Illumina protocols Naïve 2iL-I-F hESCs Elf1 NNMT KO (mutant 6.2.4) were crosslinked and chromatin processed as previously described with minor modifications. Briefly, cells were harvested with accutase and crosslinked in suspension with 1% formaldehyde solution for 10min at room temperature. Reaction was quenched with glycine and crosslinked cells were rinsed with ice-cold PBS. Nuclei were isolated and chromatin sonicated using a Covaris E210 to approximately 200-500bp size range. Magnetic Dynabeads were incubated overnight rotating at 4C with antibody against H3K27me3 (Active Motif, cat # 39155). Sonicated chromatin from approximately 200 thousand to 3 million cells was added to the bead-bound-antibodies and allowed to incubate at 4C rotating overnight. Beads were washed to remove unbound chromatin. Bound chromatin was eluted from beads and reverse crosslinked overnight. Purified DNA was prepared for next-generation sequencing via end repair, A-tailing, ligation of custom Y-adapters and PCR amplification to generate final DNA library following gel size selection. DNA libraries were prepared for sequencing using standard Illumina protocols
attributes.tag: GEO Accession
attributes.value: GSM3455704