GSM8486666: Mm, forelimb, E13.5, wt, scRNA, Rep2; Mus musculus; RNA-Seq
For single-cell gene expression analysis, mouse and bat embryonic limb tissues were dissected and dissociated with trypsin. Cells were filtered through a 40 µm Flowmi Cell Strainer (Merck, #BAH136800040) and pelleted at 300 x g at 4°C for 5 minutes. Cells were resuspended in 1 volume 0.04% BSA/PBS and dehydrated by slowly adding 9 volumes of 100% methanol. Samples were stored at -80 °C until library preparation For rehydration, dehydrated cells were centrifuged at 1000 x g at 4°C for 10 minutes and washed twice in 1 mL of rehydration buffer (1 % BSA, 0.4 U/µL Ambion RNse Inhibitor (Invitrogen, #AM1682) and 0.2 U/µL SUPERaseIn RNase Inhibitor (Invitrogen, #AM2696) in 1x DPBS). After the second wash, cells were resuspended in rehydration buffer, counted and diluted to a concentration of 1,000 cells/µL. Single cell gene expression libraries were prepared using the 10X Genomics Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10X Genomics, #PN-1000121) according to the manufacturer's instructions. The aimed target cell recovery in each experiment was 10,000 cells.  To generate Gel Beads-in-emulsion (GEMs), reactions were assembled in a Chromium Next GEM Chip G (10X Genomics, #PN-1000121). Chips were run on a Chromium Controller X/iX. Sample indices were added to the cDNAs via PCR using the Single Index Kit T Set A (10X Genomics, #PN-1000213) or Dual Index Kit TT Seat A (10X Genomics, #PN-1000215). Library concentration was measured by Qubit dsDNA HS Assay Kit (Invitrogen, #Q33231) and quality was assessed using Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent, #5067-4626). Finally, scRNA-seq libraries were sequenced on a Illumina NovaSeq 6000 with asynchronous 28 bp / 90 bp paired-end reads. Single-cell experiments were performed in biological duplicates.