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Sample GSM1223564

Query DataSets for GSM1223564

Status

Public on May 29, 2014

Title

mouse_tgc_3seq_rep2

Sample type

SRA

Source name

Trophoblast giant cells - cultured

Organism

Mus musculus

Characteristics

strain: 129/Sv
cell type: cultured trophoblast giant cells

Treatment protocol

TSCs were differentiated into trophoblast giant cells (TGCs) by using differentiation media, which consisted of TS media without FGF4, Activin, and Heparin, with the addition of retinoic acid dissolved in ethanol to the media to a final concentration of 5 μM. TSCs were differentiated for five days, replacing media every other day, before harvesting.

Growth protocol

Mouse TSCs were obtained from Dr. Janet Rossant, Hospital for Sick Children, (Toronto, Canada) and maintained in DMEM/F12 with 15 mM Hepes, 20% FBS, 2 mM glutamine, 100 ug/ml streptomycin, 1 mM sodium pyruvate, 100 uM BME, supplemented with Activin, FGF4, and Heparin following (Erlebacher et al, Developmental Biology 2004).

Extracted molecule

polyA RNA

Extraction protocol

mRNA was extracted directly from cell lysates using Dynabeads Oligo (dT)25 (Invitrogen).
500 ng of mRNA was then used to prepare 3’ RNA-Seq libraries as previously described (Beck et al, PLOS One 2010). Briefly, mRNA was heat sheared for 7 minutes to produce an average fragment size range of 300-500 bp, then used to generate cDNA libraries using a custom oligo dT primer containing Illumina-compatible adapter sequence. cDNA fragments were end-repaired and ligated to standard Illumina adapters. Size-selection was performed using E-gel SizeSelect agarose gels (Invitrogen), products were PCR amplified for 15 cycles, and purified using Ampure XP beads. Library quality was assessed using Bioanalyzer and Qubit, and sequenced on the Genome Analyzer IIx.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

Cell culture 3' RNA-Seq

Data processing

Basecalls performed using CASAVA version 1.5
3' RNA-seq reads were aligned to mm9 or rn4 using Bowtie 0.12.7 with the settings --best --strata -k 10 -S --chunkmbs 190
Using Unipeak v1.0, Bowtie alignments were filtered by posterior probability (sam_map.pl from Unipeak), and transcription peaks were called using default settings.
Peaks (chromosomal coordinates) were associated with Ensembl (e67) gene database for mouse and multiple peaks for each gene were pooled.
Read counts were normalized across libraries using DESeq
genome build: = mm9
supplementary files format and content: txt files contain pooled gene x normalized tag count

Submission date

Sep 03, 2013

Last update date

May 15, 2019

Contact name

Roberta L Hannibal

E-mail(s)

robertah@stanford.edu

Organization name

Stanford University

Department

Genetics