GSM8280382: Wildtype plate 141; Mus musculus; RNA-Seq
Genetically manipulated embryos were dissected in ice-cold 1×PBS, followed by imaging in a drop of DMEM medium supplemented with 10% foetal bovine serum (FBS) under a Nikon Eclipse Ti2 inverted microscope. Embryos were then rinsed in ice-cold 1×PBS, transferred into an Eppendorf tube containing 35 µl 0.25% Trypsin/0.02% EDTA Solution A, and incubated at 37 °C for 5 min with 2-3 times pipetting. Trypsinization was stopped by adding 10% FBS/DMEM medium. Cell suspensions were filtered through a 35 µm strainer before sorting. Single-cell cDNA libraries were prepared using the MARS-Seq method as described (Keren-Shaul et al., 2019). Briefly, reverse transcription was performed in individual wells of the sorted 384-well plate to get complementary DNA (cDNA). Subsequently, all liquid from each 384-well plate was pooled and converted to double-stranded DNA, transcribed using T7 RNA polymerase, and fragmented. A unique plate adaptor was added to each library to be sequenced together, and RNA::ssDNA (plate adaptor) ligation was performed. The purified ligates were applied to a second RT step with a primer complementary to the ligated adaptor, and the library is completed by cDNA enrichment through PCR amplification. Library concentration and profile were evaluated before sequencing. 10x ex utero: Cultured embryos were prepared for sequencing at different developmental stages after two days in culture: late streak and early head fold, assessed by light microscopy. Six similar embryos were selected for each developmental stage, pooled and dissociated using Trypsin-EDTA solution A 0.25% (Biological Industries; 030501B) for 5 minutes at 37℃. Trypsin was neutralized with media including 10% FBS and cells were washed and re-suspended in 1x PBS (calcium and magnesium free) with 400 μg/ml BSA. Cell suspension was than filtered with a 100 μm cell strainer to avoid cell clumps. Single cell viability was determined by trypan blue staining, before being diluted to a final concentration of 1000 cells/μl. Single-cell RNA-seq libraries were generated for each pool of embryos separately using 10x Genomics Chromium v3 system (5000 cell target cell recovery) and sequenced on Illumina NovaSeq 6000 platform according to the manufacturer's instructions.