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Sample GSM4104496

Query DataSets for GSM4104496

Status

Public on Feb 23, 2021

Title

small RNA-seq in the Ago2 deletion mESC, biological replicate 2

Sample type

SRA

Source name

mouse embryonic stem cells

Organism

Mus musculus

Characteristics

cell type: Ago2 deletion mESCs
genetic background: ES-E14TG2a

Growth protocol

All the mESCs used in this study were cultured on 0.5% gelatin-coated tissue culture plates in either the 15% + Lif medium (DMEM/F-12 supplemented with 15% FBS, 2mM L-Glutamine, 0.1mM MEM NEAA, 1% Penicillin-Streptomycin, 0.1 mM β-mercaptoethanol and 1000 U/mL mLIF) or the 2i + Lif medium (DMEM/F-12, 2% FBS, 2mM L-Glutamine, 0.1mM MEM NEAA, 1% Penicillin-Streptomycin, 0.1 mM β-mercaptoethanol and 1000 U/mL mLIF, 1 x N2N27, 3 uM CHIR99021 and 1 uM PD0325901).

Extracted molecule

total RNA

Extraction protocol

Total RNA were isolated from mESCs via Trizol reagent
RNA-seq library was constructed via the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420S). The small-RNA-seq libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illuminia (Set 1) (E7300S). The Trim71 CLIP-seq was performed using the previously established HITS-CLIP protocol (Moore et al., 2014).
CLIP-seq, RNA-seq, small RNA-seq

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 4000

Description

small RNA
small RNA-seq in the Ago2_deletion mESC, biological replicate 2, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv

Data processing

Basecalls performed using CASAVA version 1.4
For RNAseq and CLIPseq, sequenced reads were trimmed for adaptor sequence, then mapped to mm10 whole genome using hisat2 with parameters--mp 6,2. The resulting sam file is converted into bam file using samtools. The uniquely mapped reads were isolated using samtools.
For small RNAseq, sequenced reads were trimmed for adapter sequence using cutadapt. The resulting reads with a length between 17 and 30nt were retained for mapping by HISAT2 (v2.1.0).
The differentially expressed and non-differentially expressed miRNAs were identified by EdgeR using the Negative Binomial Generalized Linear Models with Quasi-Likelihood Tests.
The CLIPseq peak calling were performed by the Piranha, iCount, and CLIPper. The common peaks from these 3 programs were retained as Trim71-binding regions
Genome_build: mm10
Supplementary_files_format_and_content: csv files containing miRNA expression levels (in TPM) from the smallRNA-seqs, gene expression levels (in TPM) from the RNAseqs , and Trim71-binding regions from the CLIPseq

Submission date

Oct 01, 2019

Last update date

Feb 23, 2021

Contact name

Wenqian Hu

E-mail(s)

[email protected]

Phone

507-284-2133

Organization name

Mayo Clinic