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Sample GSM976927

Query DataSets for GSM976927

Status

Public on Jan 30, 2013

Title

Hairpin B-treated mESCs D0

Sample type

SRA

Source name

mESC E14

Organism

Mus musculus

Characteristics

strain: 129/Ola
cell type: mESC
age: embryonic
treatment: Hairpin B-treated
time: D0

Treatment protocol

shRNAs directed against Braveheart, AK143260, and control hairpins including AK085506 were cloned into pLKO.1 vector. Lentiviral particles were produced in 293 cells using standard protocols. ESCs were transduced with viral supernatant containing 8ug/ml of polybrene and selected after 24 hours using 1.5 ug/ml of Puromycin.

Growth protocol

Embryoid body (EB) formation: ESCs were pre-plated to remove feeders, diluted to 100,000 cells/ml in complete growth medium lacking LIF and plated on low-attachment cell culture plates (Corning) to induce aggregation. Cardiomyocyte differentiation: Mouse ESCs were cultured in feeder-free conditions using standard techniques. For directed differentiations (Kattman et al., 2011), mouse ES cells were aggregated into embryoid bodies (EB) and cultured at 75,000 cells/ml for two days in serum free media (3 parts IMDM: 1 part Ham’s F12 0.05% BSA, 2 mM GlutaMax, B27 supplement, N2 supplement) supplemented with 50 ug/ml ascorbic acid and 4.5 x 10-4 M monothioglycerol. Embryoid bodies were dissociated and reaggregated for 40 hours in the presence of 5 ng/mL human VEGF and human Activin A and human BMP4 at concentrations empirically determined depending on lot. EBs were dissociated and plated at 470,000 cells/cm2 in StemPro-34 supplemented with 5 ng/mL VEGF, 10 ng/mL human basic FGF and 25 ng/mL FGF10.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using TRIzol® Reagent according the manufacturer’s instructions. Sequencing libraries were prepared according to Illumina RNA-Seq library kit with minor modifications. Briefly, mRNA was isolated using Dynabeads® mRNA Purification Kit (Invitrogen) followed by fragmentation (Ambion) and ethanol precipitation. First and second strand synthesis were performed followed by end repair, A-tailing, paired end adapter ligation and size selection on a Beckman Coulter SPRI TE nucleic acid extractor. 200-400 bp dsDNA was enriched by 15 cycles of PCR with Phusion® High-Fidelity DNA Polymerase (NEB) followed by gel purification of ~250 bp fragments from the amplified material. Amplified libraries were sequenced on an Illumina GAIIx or HiSeq2000 sequencer, alternatively.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Illumina Offline BaseCaller1.9.3 software used for basecalling.
Sequence reads were mapped to mm9 whole genome using Tophat 1.14/ Bowtie 0.12.7 with the following parameters --min-anchor-length 6 --splice-mismatches 0 --min-intron-length 10 --max-intron-length 1000000 --min-isoform-fraction 0.0 --max-multihits 50 --no-novel-juncs --library-type fr-unstranded
Gene expression was calculated Reads per Kilobase of modeled exon per Million mapped reads (RPKM) using Cufflinks 1.3.0
Genome_build: mm9
Supplementary_files_format_and_content: A tab-delimited text file that includes RPKM values for each Sample, and fold-changes (with an added pseudo-count of 1) for matched hairpin/scrambled samples at each time-point.

Submission date

Jul 25, 2012

Last update date

May 15, 2019

Contact name

Laurie A Boyer

E-mail(s)

lboyer@mit.edu

Phone

617 324-3335

Organization name

Massachusetts Institute of Technology