e3
Homo sapiens
Protocols: 3 µg of RNA was treated with TURBO DNase according to manufacturer's instructions followed by RNeasy Minelute RNA cleanup kit and RNA quality was confirmed using Agilent 2100 Bioanalyzer with RNA Pico 6000 kit (RIN values >8). Per RNA-seq library, 1 µg of DNAse-treated RNA was treated with RiboZero Gold (Human/Mouse/Rat) kit to remove rRNAs, followed by RNA cleanup using the RNeasy Minelute RNA cleanup kit. Sequencing libraries were prepared using TruSeq Stranded total RNA LT kit according to manufacturer's instructions using 11 cycles of PCR followed by purification with Agencourt Ampure XP beads. Libraries were evaluated on an Agilent 2100 Bioanalyzer using the DNA 1000 kit and DNA concentration was measured using a Qubit dsDNA HS Assay Kit. Samples were sequenced using a HiSeq2500 instrument to generate 50-nt single-end reads, sequencing depth was 20-40 Mio reads per library. H9 human ESCs (H9 line) were cultured in mTeSR1 medium on Matrigel-coated plates. Colonies were passaged for maintanence by Gentle Cell Dissociation Reagent Primitive streak differentiation was induced by dissociation of colonies into single cells using accutase and subsequently seeding them on Matrige-coated 6-well plates containing mTESR1 medium supplemented with ROCKi at 100% confluency. Next day cells were washed with PBS, and the medium was changed to RPMI1640 medium supplemented with L-Glutamine, B27 without insulin, 10µM ROCK inhibitor and 10µM CHIR99021. For trophoblast directed differentiation human ESCs were passaged using 0,25% Trypsin-EDTA and plated as single cells (105,000 cells/cm2) on MG coated plates in DMEM/F12 medium, supplemented with 20% KnockOut Serum Replacement, Glutamax, nonessential amino acids, beta-mercaptoethanol, and 50 ng/ml BMP4. Medium was changed daily. RNA-seq
ENA first public
2019-02-25
ENA last update
2016-09-20
External Id
SAMEA4444221
INSDC center alias
Institute of Molecular Life Sciences and Swiss Institute of Bioinformatics
INSDC center name
Institute of Molecular Life Sciences and Swiss Institute of Bioinformatics
INSDC first public
2019-02-25T17:03:10Z
INSDC last update
2016-09-20T14:52:15Z
INSDC status
public
Submitter Id
E-MTAB-5099:e3
broker name
ArrayExpress
common name
human
replicate
1
sample name
E-MTAB-5099:e3