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Sample GSM2687726

Query DataSets for GSM2687726

Status

Public on Jun 28, 2017

Title

E3.5_P9_Cell15_embryo6_single

Sample type

SRA

Source name

E3.5

Organism

Mus musculus

Characteristics

developmental stage: E3.5
tissue: Mouse Embryo
strain: C57Bl/6Babr
genotype: Wild Type

Extracted molecule

polyA RNA

Extraction protocol

E3.5 blastocysts were flushed from the uterus using M2 medium and the zonae removed by short incubation in Acid tyrodes solution. Embryos at E4.5 were dissected from nascent decidua. The trophectoderm was eliminated from E3.5 and E4.5 by immunosurgery (Solter and Knowles, 1975). Embryos were incubated for 15-30 min in 20% anti mouse anti serum in N2B27 (made in house), rinsed 3 times in N2B27 and transferred to 20% freshly thawed rat serum in N2B27 for 15-30 min. Lysed trophectoderm was removed manually by repeated aspiration using a mouth-controlled finely drawn Pasteur pipette just bigger than the ICM. Isolated ICMs were placed individually in 20 µL drops of AccutaseTM in a bacteriological dish for at least 5 min until they assumed a blackberry-like appearance. They were transferred to similar sized drops of M2 and aspirated repeatedly using a very finely drawn Pasteur pipette to separate the cells. Cells were immediately placed individually into wells of lysis buffer and frozen. E5.5 embryos were dissected manually and were observed to be early E5.5 cells lacking distinct thickening of the visceral endoderm (pre-AVE migration). The extra embryonic ectoderm was removed using the tip of a pulled Pasteur pipette. The epiblast was separated from overlying visceral endoderm by aspiration, distal end first, with a pulled Pasteur pipette with diameter just bigger than the epiblast. Both epiblast and visceral endoderm were separately disaggregated and plated into wells as described above for ICMs. E6.5 embryos were visually staged as early streak (Downs and Davies, 1993) and dissected from decidua in PBS and placed into M2 solution to remove extra embryonic tissues. Differences in dissection may contribute to different proportions of primitive streak and epiblast cells obtained at E6.5. The embryonic ectodermal portion of the early streak embryo was dissociated in drops of AccutaseTM at room temperature and upon partial digestion was fully dissociated by repeated aspiration using a very finely drawn, mouth controlled, siliconised glass needle. Cells were assigned as single, doublets or multiples by visual inspection and placed directly into lysis solution and frozen prior to library generation.
mRNA from isolated single cells was isolated and amplified using the bead based capture and amplification previously described in the G&T-seq protocol which incorporates SMARTSEQ2.
Multiplexed sequencing libraries were generated from cDNA using the Illumina Nextera XT protocol and 100bp paired end sequencing was performed on an Illumina Hiseq2500 instrument.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

G&T-Seq - (SMARTSEQ2)

Data processing

Libraries were sequenced with 2x100bp paired-end reads on a HiSeq2500 platform.
Reads were trimmed using Trim Galore v0.4.3 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) using default parameters to remove the standard Illumina adapter sequence. They were mapped to the mouse GRCm38 genome assembly using HISAT2 v2.0.5. BAM files were imported to Seqmonk v1.37.0 (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). Raw read counts per transcript were calculated using the RNA-Seq quantitation pipeline.
Genome_build: GRCm38
Supplementary_files_format_and_content: The Quantitated RNA-Seq report file is tab delimited and contains the following columns: (1) mRNA name (Ensembl); (2) Chromosome; (3) Start; (4) End; (5) Strand; (7) mRNA ID (Ensembl) (8) Gene ID (Ensembl) (9) Description (Ensembl); (10) Type; (11) Orientation; (12) Distance; (13-end) raw counts

Submission date

Jun 28, 2017

Last update date

May 15, 2019

Contact name

Felix Krueger

E-mail(s)

fkrueger@altoslabs.com

Organization name

Altos Labs

Department

Bioinformatics