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Sample GSM4075616

Query DataSets for GSM4075616

Status

Public on Apr 07, 2020

Title

CDKN2A_E8.5_1b

Sample type

SRA

Source name

whole embryo

Organism

Mus musculus

Characteristics

strain: B6D2F1 maternal strain crossed with B6/CAST F1 males
tissue: embryo
developmental stage: E8.5
genotype: Cdkn2a KO
chromium single cell 3' chemistry version: v2

Treatment protocol

B6D2F1 oocytes were isolated from 5-10 week old female mice ~12h after a hormone priming superovulation regime (5 IU PSMG and 5IU hCG delivered 46 hours afterwards) and stored in 25 ul droplets of pregassed KSOM media with 1/2 amino acids (Millipore) at 37ºC and 5%C02 . Zygotes were generated either by intracytoplasmic sperm injection of decapitated B6/CAST F1 strain sperm (RNA-seq) or natural mating with B6D2F1 males (Bisulfite-seq) and cultured to E3.5 blastoycsts prior to uterine transfer into pseudopregnant CD-1 strain females (~25-35 g). Post-implantation embryos were isolated according to the embryonic day with a 24 hour offset to accommodate developmental delay as a result of uterine transfer (see extract protocol). Experimental samples were additionally injected with a cocktail of 3-4 discrete gRNA's targeted to exons of respective KO gene and Cas9 mRNA synthesized by in vitro transcription at concentrations of 33.3 ng/ul per gRNA and 200 ng/ul for Cas9. gRNA/Cas9 injections occurred within 6-8 hours post fertilization, followed by equivalent culture in KSOM media to the blastocyst stage as the control.

Growth protocol

Preimplantation embryos were cultured in pre-gassed 25 ul KSOM supplemented with 1/2 amino acids (Millipore) under mineral oil at 37ºC and 5% CO2. After ~84 hours post-fertilization, cavitated blastocysts were transferred into the uterine horns of pseudopregnant females in clutches of 10-15 per horn.

Extracted molecule

polyA RNA

Extraction protocol

Respective gene null (KO) embryos were isolated from surrogate mice. Outermost extraembryonic tissues (yolk sac, trophectoderm derived tissues) were preserved if possible. The 7-11 embryos isolated per experiment were washed in 1xPBS/0.4%BSA, pooled without pre-selection and subjected to tissue dissociation in 200 µl TrypLE Express (Gibco) for 40-60 minutes at 37ºC, with pipetting in 5 minute intervals. The cell suspension was filtered using Scienceware Flowmi Cell Strainers, 40 µM. Cells were washed twice with 1ml 1xPBS/0.4%BSA and centrifugation for 5 minutes at 1200 rpm. The cell concentration was determined using a hemocytometer and cells were subjected to single-cell RNA sequencing (10x Genomic, Chromium™ Single Cell 3’ v2).
Single-cell libraries were generated following the manual instructions of 10x Genomic (Chromium™ Single Cell 3’ v2 or v3), with the exception of fewer PCR cycles in cDNA amplification and sample index PCR than recommended in order to increase complexity of libraries. Libraries were sequenced with a minimum of 230 Mio paired end reads and parameters as described in the manual.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

Single-cell RNA-sequencing of 11 Cdkn2a KO E8.5 mouse embryos (library b)
CDKN2A_E8.5_1ab.tar.gz

Data processing

Raw base call (BCL) files generated by Illumina sequencers were demultiplexed into FASTQ files using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("mkfastq" pipeline, version 1.3.1, default parameters).
For each experiment FASTQ files were aligned, filtered, barcode and UMI counted using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("count pipeline", version 1.3.1, default parameters). The count pipeline was run jointly on multiple sequencing runs of the same experiment. Genomic reference: "refdata-cellranger-mm10-1.2.0", prepared by 10x Genomics.
For experiments with multiple libraries, all libraries were aggregated using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("aggr pipeline", version 1.3.1, default parameters). The output of Cell Ranger "count" was aggregated and normalized to the same sequencing depth and then the gene-barcode matrices were recomputed on the combined data. Sample aggregation was evaluated with normalizing for sequencing depth differences in the libraries. Normalized UMI counts used the 'mapped' normalization mode. Analysis of the data was based on the normalized UMI counts.
Genome_build: mm10
Supplementary_files_format_and_content: For all samples there is one tar gz archive containing the gene-barcode matrices as produced by the Cell Ranger pipeline. They contain the MEX-formatted UMI count matrices of each sample, or if multiple libraries where sequence, the aggregated gene-barcode matrix. Samples names consist of genotype (KO), developmental state (E6.5, E7.5, E8.5, experiment number (1, 2) with respect to genotype and stage and, if aggregated, library number (a, b). 10x Genomics explains their MEX-formatted UMI counts as follows: "The cellranger pipeline generates a gene-barcode matrix... Supplementary_files_format_and_content: Each matrix is stored in Market Exchange Format (MEX). It also contains TSV files with genes and barcode sequences corresponding to row and column indices, respectively." More information on the formatting can be found at https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/matrices.

Submission date

Sep 12, 2019

Last update date

Apr 07, 2020

Contact name

Helene Kretzmer

E-mail(s)

kretzmer@molgen.mpg.de

Organization name

Max Planck Institute for Molecular Genetics

Department

Genome Regulation