title: GSM8152422: Mixed germ cell tumour, scRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Testis specimens were placed into cold PBS and transported on ice. Tissues were digested following the standard two step enzymatic isolation protocol. Briefly, specimens were digested with collagenase type IV for 5 min at 37 °C with gentle agitation, then shaken vigorously and incubated for another 3 min. The tubules were sedimented by centrifugation at 200× g for 5 min and washed with Hanks' Balanced Salt Solution (HBSS) before digestion with 4.5 mL 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) and 4 Kunitz unit (kU) DNase I. The suspension was triturated vigorously three to five times then incubated at 37 °C for 5 min. The process was repeated in 5 min increments for up to 15 min total. The digestion was stopped by adding FBS to a final concentration of 10%. Single testicular cells were obtained by filtering through 70 µm and 40 µm strainers to obtain single cell suspensions. The cells were pelleted by centrifugation at 600× g for 15 min and washed twice with PBS. Cell number was measured using a hemocytometer, and cells re-suspended in 1× PBS/0.4% BSA. Single cell suspensions were loaded and run on the 10x Chromium Controller using Chromium Single Cell 3′ v3.1 reagents (10x Genomics PN-1000121). Sequencing libraries were prepared following the manufacturer's instructions, using 13 cycles of cDNA amplification, followed by an input of ∼100 ng of cDNA for library amplification using 12 cycles. The resulting libraries were then sequenced on a 2 X 100 cycle paired-end run on Illumina HiSeq 2500 or Novaseq 6000 instruments.