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Sample GSM2081520

Query DataSets for GSM2081520

Status

Public on May 12, 2016

Title

tdMEFs at day 4 of reprogramming (rep 3)

Sample type

SRA

Source name

reprogrammed tdMEFs at day 4

Organism

Mus musculus

Characteristics

barcode: AGTGAG
tissue: M9 reprogrammed cells
genetic background: 129xC57BL6
replicate number: 3

Treatment protocol

M9-induced neural reprogramming was performed in M9 medium (50% Neural basal, 50% DMEM/F12/Glutamax, 1xN2, 1xB27 without vitamin A, 0.075% BSA, 0.1 mM nonessential amino acids, CHIR99021 3 mM, LDN193189 100 nM, A83-01 0.5 mM, Hh-Ag1.5 0.5 mM, retinoic acid 1 mM, SMER28 10 mM, RG108 10 mM, Parnate 2 mM, bFGF 10 ng/ml) at 5% O2 and 5% CO2 incubator at 37°C.

Growth protocol

tdMEFs were cultured in MEF medium (Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 0.1 mM nonessential amino acids, and 2 mM Glutamax) in 5 % CO2 and 20 % O2 at 37 °C.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using RNeasy Plus mini kit (Qiagen), according to manufacture instruction.
RNA-seq libraries were prepared with ovation RNA-seq system v2 kit (NuGEN). In this method, the total RNA (50 ng) is reverse-transcribed to synthesize the first-strand cDNA with a combination of random hexamers and a poly-T chimeric primer. The RNA template is then partially degraded by heating and the second-strand cDNA is synthesized using DNA polymerase. The double-stranded DNA is then amplified using single primer isothermal amplification (SPIA). SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′-end of the double-stranded DNA, after which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′-end of the primer by displacement of the existing forward strand. Random hexamers are then used to amplify the second-strand cDNA linearly. Finally, libraries from the SPIA amplified cDNA were made using the Ultralow V2 library kit (NuGEN). The RNA-seq libraries were analyzed by Bioanalyzer and quantified by QPCR (KAPA). Three RNA-seq libraries were pooled per lane of paired-end 100 bp sequencing on HiSeq 2500 instrument (Illumina).
RNA-seq with Illumina HiSeq 2500. Each sample was sequenced in two lanes on an Illumina HiSeq 2500.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

batch2_3_D4_2_AGTGAG

Data processing

Paired-end RNA-seq reads were aligned to mouse genome build "mm9" using the program Tophat 2.0.13. Gene annotation (GTF file) used was ENSEMBL version 65. Specific settings: --min-anchor=5 --segment-length=25 --no-coverage-search --segment-mismatches=2 --splice-mismatches=2 --microexon-search --mate-inner-dist=50 --no-discordant --no-mixed
Aligned reads were assigned to genes using "featureCounts" (Liao et al., 2014), part of the Subread suite (http://subread.sourceforge.net/).
Differential expression P-values were calculated using edgeR (Robinson et al., 2010), an R package available through Bioconductor. Any genes where there were not at least two samples with a CPM (counts per million) value between 0.5 and 5000 were filtered out. The remaining expression values were normalized using "calcNormFactors" in edgeR. P-values for the differential expression between samples were then calculated by edgeR, using a negative binomial distribution for gene expression. Finally, FDR (false discovery rate) for each P-value were calculated by the built-in R function "p.adjust", using the Benjamini-Hochberg method.
Genome_build: mm9
Supplementary_files_format_and_content: RNA-seq FPKM, CPM (counts per million), and raw count data, tables in Excel format

Submission date

Mar 07, 2016

Last update date

May 15, 2019

Contact name

Gladstone Bioinformatics

Organization name

Gladstone Institutes

Department

Data Science and Biotechnology

Lab

Bioinformatics Core