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Sample GSM2611348

Query DataSets for GSM2611348

Status

Public on Jun 28, 2017

Title

MTY_RNA_S3_DO16051912_HJMKLALXX_L1

Sample type

SRA

Source name

chemically-induced cells from mouse fibroblasts

Organism

Mus musculus

Characteristics

strain: Mapttm2(EGFP)Klt/J
tissue: chemically-induced cells from mouse fibroblasts

Extracted molecule

total RNA

Extraction protocol

Total RNA from each sample was isolated using the RNeasy Plus Mini Kit (QIAGEN).
A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

#1 XEN-like colony (Figure S4A), #4 XEN-like colony (Figure S4B)
processed data file: Neuronal cell seq03.xls
MTY_RNA_S3_DO16051912_HJMKLALXX_L1

Data processing

The transcriptome reads were mapped using the TopHat2 program.
The gene expression level was calculated by using FPKM method.
Genome_build: mm10
Supplementary_files_format_and_content: fpkm_tracking files including FPKM values for the analysis of gene expression

Submission date

May 10, 2017

Last update date

May 15, 2019

Contact name

Yan-Tao Ma

E-mail(s)

yantao.ma89@live.com

Organization name

Peking University

Street address

No. 5 Yiheyuan Road