title: 3600STDY6184029
organism: Homo sapiens
description: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-07-26T17:02:31Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-05-08T16:32:45Z
attributes.tag: External Id
attributes.value: SAMEA4651746
attributes.tag: INSDC center name
attributes.value: Cancer Research UK Cambridge Institute
attributes.tag: INSDC first public
attributes.value: 2018-07-26T17:02:31Z
attributes.tag: INSDC last update
attributes.value: 2018-05-08T16:32:45Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6819:3600STDY6184029
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: H9
attributes.tag: cell type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: doxycycline-inducible expression of NANOG and KLF2
attributes.tag: phenotype
attributes.value: primed
attributes.tag: sample name
attributes.value: E-MTAB-6819:3600STDY6184029
attributes.tag: scientific_name
attributes.value: Homo sapiens