title: GSM2027519: D8 RNA-seq R2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was isolated using qiagen total RNA isolation kit. 500 ng of total RNA from each sample was used for ribosomal RNA removal follow RiboZero kit instructions. The resulting RNA was fragmented with divalent cations under elevated temperature. First-strand cDNA synthesis produced single-stranded DNA copies from the fragmented RNA by reverse transcription. After second-strand cDNA synthesis, the double-stranded DNA underwent end repair, and the 3′ ends were adenylated. Finally, universal adapters were ligated to the cDNA fragments, and 10 cycles of PCR was performed to produce the final sequencing library. Five to ten nanogram of MBD purified DNA was end-repaired, a single adenosine was added and ligated to Illumina adaptor oligonucleotides (TruSeq DNA sample prep Kit, Illumina). Both RNA-seq and MBD-seq library templates were prepared for sequencing using cBot cluster generation system (Illumina) with TruSeq PE Cluster V3 Kit. The sequencing run was performed with paired end mode of 100 cycles of read 1, 7 cycles of index read, and 100 cycles of read 2 using Illumina HiSeq 2500 platform with TruSeq SBS V3 Kits. Sequencing library preparation and reactions were conducted at City of Hope’s integrative genomics core