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Sample GSM3090519

Query DataSets for GSM3090519

Status

Public on Jan 01, 2023

Title

KD_random rep1

Sample type

SRA

Source name

Embryonic stem cell

Organism

Mus musculus

Characteristics

strain: 12910la
genotype: wild type

Treatment protocol

The shRNA of MuERVL was cloned into pSuper puro vector .WT mESCs were transfected with 1000ng pSuper puro vector . After transfection for 24h,the cells were treated with 1ug/ml puromycine for 4days and the medium were changed every 24h.

Growth protocol

Mouse E14 embryonic stem cells (ESCs) were cultured under 5% CO2 at 37℃ on well plate coated with 0.2% gelatin(Sigma),in medium(Hyclone) that supplement with15% FBS（Hyclone）,10ng/ml mLIF (GeneScript)，1% Penicillin-Streptomycin (Solarbio)，1% GlutaMAX(Solarbio), 1% nonessential amino acids(Gibco), 0.1 mM β-mercaptoethanol (Sigma). .

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted with Trizon Reagen (CWBIO) and was subjected to DNase Ｉ(Thermo Scientific) for 30min
Next generation sequencing library preparations were constructed according to the manufacturer’s protocol (VAHTS mRNA-seq V2 Library Prep Kit for Illumina). The mRNA fragmentation and priming was performed using VAHTSFirst Strand Synthesis Reaction Buffer and VAHTS Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified double-stranded cDNA（byAxyPrep Mag PCR Clean-up (Axygen)was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp (with the approximate insert size of 300 bp) were recovered. Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

Illumina RTA software used for image identification and basecalling.
Adaptor sequence and low-quality sequence were trimmed by Trim Galore, then sequence reads were mapped to mm10 genome using Hisat2 with default parameters.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Normalized count were calculated using a protocol from Davide Risso et al., Nature biotechnology, 2014.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file include Normalized count values for each Sample.

Submission date

Apr 09, 2018

Last update date

Jan 01, 2023

Contact name

Weiyu Zhang

E-mail(s)

ch8316f5eyu@gmail.com

Phone

18902012072

Organization name

Nankai University