GSM9149799: KO Raldh2 rep1; Mus musculus; RNA-Seq
Placentas from E9.5 embryos for each experimental condition were pooled and enzymatically digested into a single cell suspension with 1mL of 10% Collagenase type I from Clostridium histolyticum with agitation at 37°C for 1hour. Cells were passed through a 70μm cell strainer and rinsed with 1 mL ice-cold HBSS. Placentas were passed through a 70μm cell strainer into 1X red cell lysis buffer and incubated for 10 minutes at room temperature. The sample was depleted of CD45+ cells and enriched for CD31+ cells to increase the number of ECs using microbeads. 5% of all other cells were spiked back to the final sample. Single-cell suspensions were loaded into a multi-channel microfluidic chip within the Chromium Controller (10x Genomics) to generate GEMs, targeting a recovery of 5,000–10,000 cells. The 10x Genomics Chromium Single Cell System was used to process the suspension. Gene expression libraries were prepared with the Chromium™ Next GEM Single Cell GEM-X 3' kit. Sequencing was performed on the Illumina NextSeq 2000 instrument using Illumina P2-100 kit (WT and KO samples) and P4-100 kit (WT + ATRA and KO + ATRA samples).