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Sample GSM2913932

Query DataSets for GSM2913932

Status

Public on Jan 06, 2021

Title

WDR76-/- E14 total RNA seq replicate 1

Sample type

SRA

Source name

Embryonic stem cells

Organism

Mus musculus

Characteristics

strain: E14 Wdr76-/- (CRISPR biallelic ablation of exon 11)

Growth protocol

Mouse Embryonic Stem Cells (mESC) E14 cell line (129/Ola background) (Hooper et al., 1987) were grown at 37°C, 5% CO2, 95% humidity in ES media high glucose DMEM (Invitrogen) supplemented with 15% (v/v) FBS (Gibco), 2mM L-glutamine (Gibco), 1% (v/v) non-essential amino acids (Gibco), 1X Penicillin/Streptomycin (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 1000U/mL LIF (ESG1107, Millipore), 3 µM CHIR99021 (04-0004, Stemgent), 1 µM PD0325901 (04-0006, Stemgent), sterile filtered 0.1 µm membrane, stored in 4°C for up to 1 week. E14 cells were cultured on dishes coated with 0.1% gelatin (Sigma) without feeder cells, and passaged daily in 1:3 ratio (media was changed 3 hours prior passage). Cells were harvested at 80-90% confluence, with media changed at least 3 hours prior.

Extracted molecule

total RNA

Extraction protocol

RNA libraries were prepared from ribosome-depleted RNA using the Next Ultra Directional RNA Library Kit (NEB) and Next Multiplex index oligonucleotide primers (NEB) for use with Illumina sequencing platforms. The library was prepared according the manufacturer’s instructions and amplified using 15 cycles of PCR. Indices for each library sample were chosen to minimize the Hamming distance between index reads in order to ensure proper assignment of reads to samples during sequencing. The libraries were sequenced on an Illumina HighSeq4000 at the University of Chicago Functional Genomics Core Facility.
cDNA libraries were prepared from the total RNA isolated from triplicate independent cultures of wild type and Wdr76-/- (g1-3.1 clone) E14 cells grown in parallel. Briefly, cells were passaged side-by-side at similar confluence over the course of five days. Total RNA from 4 million cells of each genotype was isolated in triplicate by Trizol-chloroform extraction and purification using RNA Clean & Concentrator (Zymo). For each sample, 2 μg of total RNA was ribosome-depleted using Ribo Zero (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

Wdr76_knockout_differential_expression_refseq.txt

Data processing

Reads were mapped to mm9 reference genome, using Bowtie2 (Langmead et al., 2009), with default parameters and SAM output
SAM files were filtered to reject unmapped reads, and converted to sorted and indexted bam files using SAMtools (Li et al., Bioinfomatics 2009)
Peaks were called relative to input using macs2 using "--broad" and "--extsize 284" parameters, and filtered for overlap with mm9-blacklist by Bedtools (https://sites.google.com/site/anshulkundaje/projects/blacklists)
align to mm9 genome with tophat2
prepare for DE analysis with cufflinks then cuffmerge
Differential Expression was performed in Cuffdiff with rRNA, tRNA, 7SK, snoRNA, and mitochondrial RNA masked using per condition dispersion fitting, then filtered for blacklist and refseq overlap using bedtools.
Genome_build: mm9
Supplementary_files_format_and_content: bed file of called ChIP peaks with fourth column normalized ChIP signal (IP/input); txt file of differential expression

Submission date

Jan 05, 2018

Last update date

Jan 06, 2021

Contact name

Alexander Ruthenburg

E-mail(s)

aruthenburg@uchicago.edu

Organization name

University of Chicago

Department

MGCB/BMB