GSM8809648: Dissociated E9.5 somite cells; Mus musculus; RNA-Seq
Pregnant C57BL/6 wild type mice were sacrificed at 9.5 days and 11.5 days p.c. Embryos were extracted and interlimb somites were dissected in a PBS, 1% BSA filled plate using Dumont #5SF forceps. Somites from three embryos of each embryonic age were aspirated with a wide-bore p200 pipette tip with the surrounding PBS and pooled together in low-bind 1.5 mL tubes. The pooled somites were centrifuged at 100g, 4°C for 2 min. Supernatant was aspirated and replaced with 50 µL trypsin (325-043-CL, Wisent Bioproducts) and incubated at 37°C for 15 min. 200 µL of fetal bovine serum (FBS, 090150 Wisent Bioproducts) was then added, and cells were dissociated by pipetting up and down. Cells were centrifuged at 200g for 4 min at 4°C. Cells were washed twice with DMEM supplemented with 10% FBS then manually counted (viability >90%) and filtered with a Flowmi 40 µm tip strainer (cat. H13680-0040, Belart) and subsequently re-counted. Cells were adjusted at 1,100 cells/µL and a 150 µL aliquot was sent for analysis Aliquot was sent for analysis using the 10X Chromium Chip (10X Genomics) for library preparation. Sequencing was performed with a High Output (150 cycles) flowcell using Illumina Nextseq 500. For each sample (E9.5 and E11.5), a total of 10,000 cells were sequenced with approximately 30,000 median reads per cell and 3,500 median genes per cell.