GSM6739287: scRNA_RSeT_induced_GATA3_AP2y_hESC; Homo sapiens; RNA-Seq
To collect post-implantation embryo models for single cell sequencing, correctly organized embryoids at day 4, 6 and 8 were picked visually and washed through PBS twice in a 4-well dish before transfer to TrypLE. Samples were agitated by pipetting every 5 minutes for 10-20 minutes until dissociated. Enzymatic activity was inactivated through addition of 20% FBS in PBS at 2x volume. Cells were collected in a falcon tube, pelleted, and resuspended in freeze buffer consisting of 50 mM Tris at pH 8.0 (15-567-027, Fisher Scientific), 25% glycerol (G5516, Sigma-Aldrich), 5 mM Mg(OAc)2 (63052, Sigma-Aldrich), 0.1mM EDTA (15575020, ThermoFisher Scientific), 5mM DTT (R0861, ThermoFisher Scientific), 1x protease inhibitor cocktail (P8340, Sigma-Aldrich), 1:2500 dilution of superasin (AM2694, Invitrogen). For cell lines, 10,000 cells were counted, pelleted, resuspended in the freeze media above, snapped frozen in liquid nitrogen then stored at -80*C. For nuclei isolation and library construction, low input nuclei isolation protocol from 10x Genomics was performed. Briefly, frozen cell pellets were thawed in a 37°C water bath for 30 seconds, centrifuged (500g for 5 minutes at 4°C) to pellet the cells then the supernatant were aspirated. The cell pellets were washed with 200 ul 1x PBS with 0.04% BSA twice, centrifuged and aspirating the supernatant in between washes. Then, chilled lysis buffer (45 ul per sample) was added to the washed cell pellet and placed on ice for 3 minutes, then wash buffer (50 ul per sample) was added. Washed isolated nuclei were resuspended in a diluted nuclei buffer. In this study, the isolated nuclei were resuspended in 5 ul of diluted nuclei buffer and were directly added to the transposition reaction. All the steps proceeding, 10x Genomics' Single Cell Multiome ATAC and Gene expression protocol were followed to manufacturer's specifications and guidelines. The final libraries were loaded on the NextSeq 2000 using P2 100 cycle kit at 650 pM loading concentration with paired end sequencing following the recommended sequencing reads from 10x Genomics (28/10/10/90 cycles for gene expression libraries and 50/8/24/49 cycles for ATAC libraries). 