title: GSM7431433: FTW-mESCs/FTW-mTSCs/FTW-mXENs Co-culture 2; Mus musculus; RNA-Seq
library_construction_protocol: Mouse blastocysts were collected at E3.5 and the zona pellucida removed by Tyler's buffer as described previously(Yu et al., 2021). After removing the zona, blastocysts were washed in PBS/PVA two times. Digestion was performed in TrypLE Express for 10 minutes at 37 ℃, and cell clumps were cut up by mouth glass pipettes beginning with a larger size (50 um diameter) to a smaller size (20 um diameter). The totally digestion time was around 40-50 minutes. For the FTW-derive-day 8 outgrowth samples, blastocysts were collected and plated onto MEF-coated plates in FTW medium for 8 days, and then the outgrowth area were picked and dissociated.  For the established FTW cell lines, all the cells were dissociated with TripLE Express for 3-5 minutes at 37 ℃. Single cell suspensions were diluted at a concentration of 1100 cells/µL in 0.04% bovine serum albumin (BSA)/PBS for loading into 10X Chromium Single Cell G Chips (for mouse blastocysts, all the of the cells were loaded into the chip). Single cell libraries were prepared using the Chromium Single Cell 3' Reagent Kit v3.1 (10X Genomics, Pleasanton, CA) following the manufacturer's protocol. Briefly, single cells were partitioned into Gel beads in EMulsion (GEMs) in the GemCode instrument followed by cell lysis and barcoded reverse transcription of RNA, amplification, shearing and 5' adaptor and sample index attachment. Libraries were sequenced on the Illumina NovaSeq 6000 platform.