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Sample GSM3272281

Query DataSets for GSM3272281

Status

Public on Aug 20, 2019

Title

Wild Type1

Sample type

SRA

Source name

Embryonic stem cell

Organism

Mus musculus

Characteristics

strain: 12910la
genotype: wild type

Treatment protocol

ESCs were transfected 0.5 μg Kdm2a gRNA using 3 μl Polyjet (Signagene, SL100688). 2-3 days later, 1.0 × 104 GFP+ cells were sorted by flow cytometry and single ESC clone was expanded for establishing Kdm2a-/- cell line.

Growth protocol

ESCs were cultured in medium containing DMEM/HIGH GLUCOSE (HyClone, SH30243.01), 15% FBS (HyClone, SH30070.03), 1X MEM NEAA (Gibco, 11140-050), 2 mM L-Glutamine (Solarbio, G0200), 100 U/ml penicillin, 100 µg/ml streptomycin (Solarbio, P1400), 0.11 mM 2-Mercaptoethanol (Sigma, M3148-250ml) and 10 ng/ml mouse LIF (GeneScript, Z03077) on 12 well tissue culture plates (FALCON, 353043) coated with 0.2% gelatin (SIGMA, G1890-100G).

Extracted molecule

total RNA

Extraction protocol

Total RNA of cells were extracted by Trizon reagent (CWBIO,CW0580) following the manufacture‘sprotocol. For genes expression analysis, total RNA was treated by DNase I (Thermosentific, EN0521) for 30~45min to remove genome DNA. DNase I was inactivated in 70~75 °C for 10min.
Next generation sequencing library preparations were constructed according to the manufacturer’s protocol (BGISEQ-500 RNA-Seq (Quantification) Library Preparation Protocol). mRNA were fragmented into small pieces using fragmentation reagent. First-strand cDNA was generated using random hexamer-primed reverse transcription , then was followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3’ adenylated. Adaptors were ligated to the ends of these 3’ adenylated cDNA fragments. PCR is to amplify the cDNA fragments with adaptors from previous step. PCR products are purified with the SPRI beads, and dissolved in EB solution. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library . Library was validating on the Agilent Technologies 2100 bioanalyzer.
Library was prepared with BGISEQ kit and sequenced on BGISEQ-500 with 50bp read length for 24 million reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Data processing

Adaptor sequence and low-quality sequence were trimmed by SOAPnuke, then sequence reads were mapped to mm10 genome using Hisat2 .
Reads count were caculated by FeatureCounts.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file include count values for each Sample.

Submission date

Jul 16, 2018

Last update date

Aug 20, 2019

Contact name

Yongwang Zhang

E-mail(s)

zyw_ujn_nku@foxmail.com

Organization name

Nankai University

Department

The State Key Laboratory of Medicinal Chemical Biology