title: GSM3617777: F03-3iN RNA-Seq; Homo sapiens; RNA-Seq
library_construction_protocol: Genomic DNA was extracted from fibroblast and iPSC's using Blood & Cell Culture DNA mini kit (Qiagen) and iN's using Quick –DNA Miniprep Plus Kit (Zymo Research).  Total RNA was extracted from fibroblast, iPSC's and iN's using the RNeasy mini kit and RNeasy micro kit (Qiagen). Libraries were prepared according to KAPA biosystems instructions accompanying KAPA library Preparation Kit Illumina platforms. In brief, genomic DNA was sonicated (Covaris) to generate fragments 180-220 bp in size. Following fragmentation, DNA was used to construct library. After library construction, DNA libraries were bisulfite converted and purified using the EZ DNA Methylation Lightning Kit (Zymo Research). Bisulfite converted samples libraries were amplified using LM-PCR. Amplified bisulfite converted samples and SeqCap Epi probes were hybridized for 64-72 h. Captured DNA samples were washed and recovered, followed by amplification of the capture DNA samples using LM-PCR. Amplified samples were purified using Agencourt AMPure XP Beads (Beckman Coulter). Bisulfite converted libraries captured by SeqCap Epi CpGiant Probes kit sequenced on Illumina HiSeq 4000 platform by 2x150 paired-end.   For RNA-seq, library construction was performed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) following manufacturer's instructions. Briefly, total RNA was converted to mRNA using polyA purification. The mRNA was then fragmented and primed for cDNA synthesis. The cDNA was adenylated and adapters were ligated onto the cDNA. Samples were amplified using PCR, amplified library was then purified using Agencount AMPure XP beads (Beckman Coulter). Final libraries were checked for quality on a Bioanalyzer DNA High Sensitivity chip (Agilent). Stand-specific RNA libraries of all the samples were sequenced on Illumina NextSeq 550 platform using 2x150 paired-end sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3617777