Illumina HiSeq 2500 paired end sequencing; GSM2687696: E3.5_P11.P6.P10_cell181_embryo_single; Mus musculus; RNA-Seq

E3.5_P11.P6.P10_cell181_embryo_single

This study explores lineage and regulatory processes involved in early post implantation mouse embryos using single-cell RNA-seq Overall design: Single cells from C57Bl/6Babr  mouse embryos at E3.5, E4.5, E5.5 and E6.5 were isolated and subjected to single-cell RNA-seq protocol.

Single-cell landscape of transcriptional heterogeneity and cell fate decisions during mouse early gastrulation

Submitted by Gene Expression Omnibus on 29-JUN-2017

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 ÂµL 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.

Single-cell landscape of transcriptional heterogeneity and cell fate decisions during mouse early gastrulation

Submitter Supplied Information

Samples