title: GSM2876149: TERT+ OCT4-GFP+ gonocytes from P6 Rep 4; Mus musculus; RNA-Seq
library_construction_protocol: Testes were dissociated based on standard two-step protocols (Brinster and Zimmermann, 1994). Briefly, testes were removed and detunicated using sharp forceps, and placed into HBSS (137mM NaCl, 5.4mM KCL, 0.44mM KH2PO4, 0.25mM Na2HPO4, 4.2mM NaHC03, 1G/L glucose, 9mM HEPES ph7.4) + 2% fetal bovine serum. Tubules were placed into 5ml/testis of 1mg/ml collagenase IV (Worthington) for 5 min at room-temperature for the removal of interstitial cells. Detangling was assisted using sharp forceps as well as trituration with a bulb pipette. The tubules were collected in a 100um filter basket, washed with HBSS, and incubated for 15min at 32C in 5ml/testis of 0.25% trypsin (Invitrogen; EDTA-free) + 1mg/ml collagenase IV + Dnase1 (Invitrogen), with frequent trituration with a bulb pipette. The cell preparation was filtered through a 40um basket, and in some experiments untrypsinized cell were subjected to additional 10min room-temperature incubation in HBSS/FBS + 5mM EDTA.  The two dissociation fractions were washed and pooled. Cells were sorted directly into Trizol LS (Invitrogen), and RNA extracted following the manufacturer's recommendation. RNA was further purified on Rneasy Micro columns (Qiagen). cDNA was prepared and amplified using the Nugen Ovation V2 kit, starting from 5 to 10ng of total RNA. The cDNA was sonicated to 200bp size using a Covaris S2 machine, and 25ng of cDNA was used to make the libraries, following standard Illumina Truseq V2 protocols. Samples were sequenced on an Illumina Hiseq2500 machine, with paired-end 101bp reads.