GSM7610831: BVSC Gastruloid, 96h, STELLA+, rep1; Mus musculus; RNA-Seq
Gastruloids were collected and washed twice in PBS before incubating at room temperature for 8 mins in Trypsin-EDTA before quenching with 10% FBS in PBS. Cells were pelleted at 230xg for 5mins before resuspending in filtered 1% FBS in PBS. Cell solution was passed through tube filter (35µm) then counted and divided into tubes before antibody addition. Incubated at 4°C on rotator for 1 hour then centrifuged at 800 rpm for 5 mins at 4°C. Supernatants were aspirated and sample washed with filtered 1% FBS in PBS before repeating wash and transferring to chilled flow tubes. Cells were loaded onto a BD FACSAria™ Fusion III (BD Biosciences) for sorting. Sorting was based on the reporters Stella::CFP, Blimp1::mVenus or PECAM1-PE and SSEA1-A647 antibodies. Sorted cells were transferred to DNA low-bind tubes and centrifuged at 300 rcf for 5 mins at 4°C. The supernatant was aspirated and, using cut tips, 1ml of chilled PBS pipetted up and down 10 times. This was repeated twice more, and after the final centrifugation step the cells were resuspended in 200µl chilled PBS then 800µl of chilled 100% methanol added dropwise and with stirring. Fixed cells were stored at -80°C until ready for 10x preparation for scRNAseq. The sorted, fixed and frozen cells were thawed on ice for 5 minutes before centrifugation at 1,000 xg for 5 minutes (at 4°C). Supernatant was carefully aspirated without disturbing the pellet before resuspending the pellet in the appropriate volume of Wash-Resuspension buffer; 3x SSC Buffer (Invitrogen, 15557-044) supplemented with 0.04% Bovine Serum Albumin (BSA) (Invitrogen, AM2616), 1mM DL-Dithiothreitol solution (DTT) (Sigma-Aldrich, 646563) and 0.2U/µl Protector RNase inhibitor (Roche, 3335399001) to give 1000 cells/µl in 50µl or minimum volume of 50µl if not possible to obtain that concentration. Quality control on the cells and counts were performed on a Luna FX7 cell counter (Logos biosystems) prior to applying to 10x Chromium library preparation performed according to manufacturer's instructions. Single cell libraries of 100 bp paired-end reads were pooled and sequenced on HiSeq 4000 to perform single cell RNA sequencing (scRNAseq).