title: GSM4466781: Fetal - 23 wks_1; Homo sapiens; RNA-Seq
library_construction_protocol: Thymic tissues placed in RPMI containing 100 μg/ml DNase I were cut into small pieces using scissors. Tissue pieces were transferred into a gentleMACS C tube containing 10 ml of RPMI with DNAse. The gentleMACS Program m_spleen_02 was run three times. Thymic fragments were separated from the thymocytes-rich supernatant by centrifugation. Remaining fragments were transferred back to C tube with fresh RPMI with DNAse before running program m_spleen_01. The supernatant was removed and replaced with 10 ml of digestion medium containing 100 μg/ml DNase I and 100 μg/ml Liberase TM in RPMI. Tubes were moved to a 37°C water bath and fragments were triturated every 5 minutes to mechanically aid digestion. At 30 min, tubes were spun briefly to pellet undigested fragments and the supernatant was discarded. Fresh digestion medium or accumax was added to remaining fragments and the digestion was repeated for another 15-30 minutes until most pieces were digested. Supernatant from this second round of digestion was transferred to a tube containing cold MACS buffer (0.5% BSA, 2 mM EDTA in PBS) to stop the enzymatic digestion. If necessary, a third round of enzymatic digestion was performed on remaining fragments using accumax for an additional 5-10 min. Cells were pooled with the supernatant from the previous round of digestion and were passed through a 40 μm filter (Falcon). Some samples were treated with 2 ml of ACK lysing buffer for 5 min prior to stromal enrichment. For fetal and postnatal tissues, single cells from digested tissue were resuspended in MACS buffer containing 10 μM of the ROCK inhibitor Y-27632. Human CD45 MicroBeads were used to deplete immune cells according to the manufacturer's instructions with the following modification: 5 μL of CD45 MicroBeads per 10e7 total cells were added instead of 20 μL. Stromal cells from adult thymus were enriched using Fluorescence activated cell sorting (FACS). Cells were blocked and stained for 20 min using human-specific antibodies against EPCAM and CD45. After staining, cells were washed and resuspended in FACS buffer containing DAPI. Cells were sorted on BD FACS Aria II. Pre-gating was first done for live cells based the DAPI stain. Single cells were captured using the 10X Chromium microfluidics system (10X Genomics). The cells were encapsulated and barcoded cDNA libraries were prepared using the single-cell 3' mRNA kit (v2 or v3; 10X Genomics) following the manufacturer's protocol.
attributes.tag: GEO Accession
attributes.value: GSM4466781