title: Illumina HiSeq 2000 paired end sequencing; RNA sequencing of conventional and reset human pluripotent stem cells
design: RNA sequencing of conventional and reset human pluripotent stem cells
library_construction_protocol: Cells were cultured in N2B27 medium (Ndiff227, StemCells Inc) supplemented with 1uM PD0325901 (PD03) and murine LIF (prepared in-house). Total RNA was extracted with the TRIzol/chloroform method (Invitrogen), followed by resuspension in RNAsecure (Ambion), incubation with TURBO DNase (Ambion) at 37 C for 1 hour, further phenol/chloroform extraction and ethanol precipitation. RNA integrity was assessed with the RNA 6000 Nano assay on the 2100 Bioanalyzer (Agilent). Ribosomal RNA was depleted from 5ug of total RNA using Ribo-Zero capture probes (Epicentre). RNA samples were sheared by ultrasonication on a Covaris S2 for 90s set at Duty Cycle 10, Cycles per Burst 200 and Intensity 5. Fragmented RNA was reverse-transcribed with a combination of random hexamer and oligo-dT primers (New England Biolabs) by SuperScript III (Invitrogen) at 50 C for 2h in the presence of 6ug/ml actinomycin D (Sigma) to inhibit second-strand products. Second-strand cDNA was synthesized by DNA Polymerase I in the presence of RNase H with dUTPs substituted for dTTPs at 16 C for 2h. Sequential end repair and 3'-adenylation of cDNA products was carried out with T4 DNA polymerase and T4 polynucleotide kinase (20 C), and with exo- Klenow fragment (65 C) in the presence of dATPs (New England BioLabs). These were ligated to barcoded adapters (NEXTflex-96, Bioo Scientific) by T4 DNA ligase (New England BioLabs) at 20 C for 30min. Second-strand DNA was digested with uracil DNA glycosylase (UDG) and Endonuclease VIII at 37 C for 30min. PCR amplification of first-strand library constructs was carried out with KAPA HiFi DNA polymerase (Kapa Biosystems) for 13 cycles. Purification of reaction products at each step was performed with Ampure XP paramagnetic beads (Beckman Coulter). Library size distribution and molarity was assessed by the DNA 1000 assay on the 2100 Bioanalyzer (Agilent).
attributes.tag: Experimental Factor: cell type
attributes.value: embryonic stem cell
attributes.tag: LIBRARY_STRAND
attributes.value: first strand