Homo sapiens
Protocols: Human H9-NK2 ESCs were kindly provided by Austin Smith. These cells contain NANOG and KLF2 transgenes under the control of doxycycline-dependent promoters. Naive hESCs were grown in 6-well dishes on mouse embryonic fibroblasts in N2B27 supplemented with human LIF, 1 uM Chiron, 1 uM PD03 and 2 uM Go6983. One passage before sorting, cells were plated on 6-well plates coated with Matrigel (growth-factor reduced). Primed hESCs were grown in 6-well dishes coated with Vitronectin in E8 media. A cDNA library was prepared from each sorted single cell following the SmartSeq2 protocol. Briefly, oligo-dT primer, dNTPs (ThermoFisher, cat. no. 10319879) and ERCC RNA Spike-In Mix (1:25,000,000 final dilution, Ambion, cat. no. 4456740) were added to the single-cell lysates, and reverse transcription and PCR were performed. hESCs were dissociated with Accutase and sorted with a BD Aria Cell sorter, gating for cell size and granularity. Single-cells were sorted in 2uL of Lysis Buffer (0.2% v/v Triton X-100 (Sigma-Aldrich, cat. no. T9284) with 2U/ul RNase Inhibitor (Clontech, cat. no. 2313A)) in 96 well plates, spun down and immediately frozen at -80 degrees Celsius. The cDNA libraries for sequencing were prepared using Nextera XT DNA Sample Preparation Kit (Illumina, cat. no. FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1). Libraries from 96 single cells were pooled and purified using AMPure XP beads (Beckman Coulter).
ENA-FIRST-PUBLIC
2018-07-26T17:02:32Z
ENA-LAST-UPDATE
2018-05-08T16:32:39Z
External Id
SAMEA4651426
INSDC center name
Cancer Research UK Cambridge Institute
INSDC first public
2018-07-26T17:02:32Z
INSDC last update
2018-05-08T16:32:39Z
INSDC status
public
Submitter Id
E-MTAB-6819:3600STDY6184477
broker name
ArrayExpress
cell line
H9
cell type
embryonic stem cell
common name
human
developmental stage
embryo
disease
normal
genotype
doxycycline-inducible expression of NANOG and KLF2
phenotype
primed
sample name
E-MTAB-6819:3600STDY6184477
scientific_name
Homo sapiens