H9_reset_R3
Homo sapiens
Protocols: Cells were cultured in N2B27 medium (Ndiff227, StemCells Inc) supplemented with 1uM PD0325901 (PD03), 1uM CHIR99021 and human or murine LIF (according to species) (t2iL) + 5uM PKC inhibitor Go6983 (Sigma-Aldrich). piggyBac (PB) transposon vectors (2ug) carrying doxycycline-inducible KLF2 or NANOG coupled to Venus were co-transfected with an rtTA expression construct (2ug) and pBase helper plasmid (4ug) using the Neon Transfection System (Program 14; Invitrogen). Two days later, G418 was applied (100ug/ml). After selection for two weeks, Venus-negative cells were isolated by flow cytometry to remove cells with residual transgene expression. To induce resetting, transfected PSC were dissociated with trypsin and replated in the presence of Rho-associated kinase inhibitor (ROCKi) (Y-27632, Calbiochem). Doxycycline (DOX, 1uM) was added the following day and 24h later medium was changed to N2B27 medium (Ndiff227, StemCells Inc) with 1uM PD0325901 (PD03), human LIF (prepared in-house), 3uM CHIR99021 (2iL) or 1uM CHIR99021 (t2iL) and DOX. Medium was changed daily. Cells were split every five to seven days after dissociation with Accutase (Life Technologies). 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).
ENA-FIRST-PUBLIC
2014-09-10T17:02:19Z
ENA-LAST-UPDATE
2018-03-08T21:40:16Z
External Id
SAMEA2738346
INSDC center name
European Molecular Biology Laboratory
INSDC first public
2014-09-10T17:02:19Z
INSDC last update
2018-03-08T21:40:16Z
INSDC status
public
Submitter Id
E_MTAB-2857:H9_reset_R3
broker name
ArrayExpress
cell line
H9
cell type
pluripotent stem cell
common name
human
sample name
E_MTAB-2857:H9_reset_R3
scientific_name
Homo sapiens
specimen with known storage state
fresh specimen