alias: GSM3223606
title: GSM3223606: hMSC_Luc_3 RNA-Seq; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from one million cells using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Genomic DNA extraction from cell pellets was performed according to the DNeasy Blood & Tissue Kit (Qiagen) protocol. RNA library construction protocol: The mRNA was isolated from 2 ug of total RNA using the NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB). Subsequently, the isolated mRNA was sheared, and the synthesized cDNA was used for RNA library construction using NEBNext ultra RNA library prep kit for Illumina (NEB). DNA library construction protocol: 500 ng of genomic DNA was sheared into approximately 300-bp fragments by Covaris S2. Then the DNA library was constructed using a NEBNext ultra DNA library prep kit for illumina (NEB). PBAT library construction protocol: Samples of 50 ng of genomic DNA were used to construct the PBAT library as previously described with several modifications. Briefly, together with 1% unmethylated lambda DNA (Thermo Scientific), genomic DNA was bisulfite converted and purified with EZ-96 DNA Methylation-Direct™MagPrep (Zymo) according to the manufacturer's instructions. The bisulfite-converted DNA was subjected to four rounds of random priming and extension with biotinylated random primers containing a truncated Illumina P5 adaptor and supplemented with klenow exo– polymerase (Enzymatic). The excess primers were digested by exonuclease I (NEB). After purification of DNA with 0.8× Ampure XP beads (Beckman), the newly synthesized DNA strands were bound to the Dynabeads M280 Streptavidin (Invitrogen), and the bisulfite-converted DNA templates were removed with two rounds of 0.1 N NaOH washes. Next, the second strands were synthesized with random primers containing a truncated P7 Illumina adaptor. The M280 beads were washed again and the library was amplified with KAPA HiFi ReadyMix (Kapa biosysems) for 6-8 cycles. The generated libraries were purified twice with 0.8× Ampure XP beads. All the generated libraries were pooled and sequenced on Illumina HiSeq 4000 platforms with paired-end 150-bp sequencing. RNA-Seq; whole-genome sequencing; Bisulfite-Seq
identifiers.namespace: GEO
identifiers.id: GSM3223606