GSM5114046: Sample adrenal from 14GW female1 (10x genomics); Homo sapiens; RNA-Seq
The fresh samples of human fetal adrenal glands or gonads were placed into RPMI 1640 medium (GIBCO, C11875500BT), which containing 5% Fetal Bovine Serum (FBS; HyClone, SH30070.03) and 1% penicillin-streptomycin (Gibco, 15140-122) and quickly transported to laboratory on ice. Then the fetal adrenal glands and gonads were separated under stereomicroscope (Nikon) and collected in DNA LoBind tubes (Eppendorf) with the cold RPMI 1640 solution containing 5% FBS. Then, washed in cold D-PBS (WISENT, 311-425-CL), and sliced into approximately 1-2mm3 pieces. The tissues were transferred to digestion solution (0.1mg/ml Liberase TL (Roche, 5401020001) and 1mg/ml DNase I (Roche, 10104159001) in RPMI 1640), and digested at 37 °C with gentle shaking throughout, adrenal glands for 20 min and gonads for 30 min, respectively, then filtered through a 100μm cell strainer (Falcon, 352360), centrifuged and resuspended in 5 ml of 1X red blood cell (RBC) lysis buffer (BioLegend, 420301) for 5 min on ice. The suspensions were washed twice with D-PBS containing 1% FBS, and then filtered by a 40 μm cell strainer (Falcon, 352340), centrifuged and resuspended in DPBS with 1% FBS. The cell number and viability were assessed by Trypan blue (Gibco, 15250-061) staining and counting in a counting chamber.   Except for above, some steps were adjusted to get Aorta-gonad-mesonepheros region (AGM) at 6-7 gestation weeks (GW) —AGM suspension was passed through a 70μm cell strainer directly after digestion and didn't need to lysis RBC. For 10x Genomics single-cell RNA sequencing,reverse transcription and library preparation were performed using the 10x Genomics Single Cell v3 kit following the 10x Genomics protocol. Briefly, we added the single cell suspension, gel beads and partitioning oil to 10x Genomics Chromium chip B and ran the Chromium Controller. After water-in-oil generation, samples were transferred into a PCR tube and reverse transcription was performed using T100 Thermal Cycler (Bio-Rad). Then cDNA purification and library preparation were performed as the user guide. cDNA librarys were send to company Genergy Biotech (Shanghai) and sequenced by Novaseq 6000 (Illumina).    For Smart-seq2, Single cells were acquired and picked into cell lysis buffer by mouth pipette. Transcriptomes of the single cells were amplified refer to the previous method 4,5. Briefly：We reverse transcribed RNA molecules using 25 nt oligo (dT) primer anchored with 8-bp unique molecular identifiers (UMIs). After the first-strand and second-strand cDNA were synthesized, and the cDNA were amplified by 15 cycles of PCR. Absorbed 4μL amplified cDNA for further step. The cDNAs were biotinylated pre-indexed primers to further amplify the PCR product. The product was fragmented into ~300-bp fragments using sonicator (Covaris M220) and was captured by Dynabeads-MyOne Streptavidin C1 beads (Invitrogen,65002). The RNA-seq library was constructed using a Kapa HyperPrep kit (Kapa Biosystems, kk8504). The libraries were subjected to 150 bp paired-end sequencing on an Novaseq 6000 (Illumina) platform in Genergy Biotech company (Shanghai).