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Sample GSM4213278

Query DataSets for GSM4213278

Status

Public on Aug 23, 2020

Title

Usp7_Control rep2

Sample type

SRA

Source name

E14 embryonic stem cells

Organism

Mus musculus

Characteristics

cell line: E14 embryonic stem cells
strain: 12910la
genotype: control

Treatment protocol

We used Cas9-gRNA to knockout Ssrp1 in embryonic stem cells. Wild-type (WT) ESC and Ssrp1 knockout cell lines were cultured under serum conditions. The shRNA of Usp7 was cloned into pSuper puro vector. WT ESCs were transfected with 1000ng pSuper puro vector. After transfection for 24h, the cells were treated with 1ug/ml puromycine for 3days 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 with 15% 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 RNAiso Plus (Takara) 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 Cutadapt, then sequence reads were mapped to mm10 genome using Hisat2.
Raw count were generated by featureCounts.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file include raw count values for each Sample.

Submission date

Dec 10, 2019

Last update date

Aug 23, 2020

Contact name

Weiyu Zhang

E-mail(s)

[email protected]

Phone

18902012072

Organization name

Nankai University