GSM4677803: WT1 E16.5; Mus musculus; RNA-Seq
Cell isolation of epicardial cells at E12.5 and E16.5 for scRNA-seq: EPDCs were collected from Wt1CreERT2/+ ; R26mTmG/+ embryos that were administered 4-OHT at E9.5 and E10.5 via pregnant dams. A total of 7 E12.5 staged hearts were pooled from 2 dams; and a total of 17 E16.5 staged hearts were pooled from 4 dams based on visual confirmation of green fluorescent protein (GFP) expression in the epicardium using a Zoe Fluorescent Cell Imager (Bio-rad). Hearts negative for the expression of the Wt1CreERT2 allele, exhibited tdTomato fluorescence alone and were either discarded or used as tdTomato positive fluorescent controls for flow cytometry. Developmentally timed C57BL/6J embryos were utilized for flow cytometry as non-fluorescence controls. Additionally, genomic DNA was isolated from all embryos and all visually screened Wt1CreERT2 ; R26mTmG/+ positive embryos were confirmed using transgene specific primers followed by PCR (genotyping). Following the digestion protocol describe above, EPDCs were gated as single cells (based on FSC x SSC dimensions), DAPI negative, tdTomato negative and GFP positive. TdTomato positive cells were sorted for downstream gene expression analysis. EPDCs collected via FACS were immediately processed for single cell capture and sequencing. Cell isolation of endothelial cells at E14.5 for scRNA-seq: Endothelial cells (ECs) were collected from Wt1CreERT2/+ mice (Control) and Wt1CreERT2/+ ; Mrtf-a-/- ; Mrtf-bflox/flox (MRTFepiDKO) after administration of 4-OHT at E9.5 and E10.5 via pregnant dams. A total of 10 Control hearts were pooled from 2 dams; and a total of 7 MRTFepiDKO hearts were pooled from 2 dams. Prior to digestion, hearts were placed in HBSS at 37°C and 5% CO2 and genomic DNA from all embryos was subjected to genotyping to detect the Wt1CreERT2 allele within two hours. Following confirmation of positive embryos, hearts were subjected to the digestion protocol described above. After filtering and centrifuging cells, ECs were incubated with fluorescently conjugated bodies to select for vascular cells (CD31-APC; BD Biosciences 551262) and exclude hematopoietic cells (CD45-FITC; BD Biosciences 553079) for 30 minutes in 0.5% BSA in DPBS and on ice. After antibody labeling, cells were washed and centrifuged at 200g for 5 minutes and then in 10%FBS/DMEM buffer. ECs were gated as single cells, DAPI negative, CD45-FITC negative and CD31 positive. ECs collected were immediately processed for single cell capture and sequencing. Cellular suspensions were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-in-Emulsions (GEMs). Single-cell RNA-Seq libraries were prepared using Chromium Single-Cell 3′ Library & Gel Bead Kit (10x Genomics). The beads were dissolved and cells were lysed per manufacturer's recommendations. GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken and the pooled post-GEM-RT reaction mixtures were recovered and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, PN37002D, ThermoFisher Scientific). The entire purified post GEM-RT product was amplified by PCR. This amplification reaction generated sufficient material to construct a 3' cDNA library.  Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by End Repair, A-tailing, Adaptor Ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification. Sequence data was generated using Illumina's HiSeq2500v4.
GEO Accession
GSM4677803