title: GSM6339538: Initial segment of the murine epididymis; Mus musculus; RNA-Seq
library_construction_protocol: Organ were collected and single cell suspension was prepared by mechanical separation (chopping), followed by enzymatic digestion (1.5 mg/ml collagenase D, 60 U/ ml Dnase I, 60 U/ml hyaluronidase type I-S in DMEM, 45 minutes at 37°C) and aspiration through 20G needle. Subsequently, red blood lysis was performed for 3 minutes using RBS lysis buffer (Qiagen). Cells were staining with PerCP-Cyanine 5.5-conjugated CD45.1 antibody (BioLegend, RRID: AB_893346). Single live CD45.1+CD45.2- immune cells (extravasular CD45+ cells) were sorted using an BD Aria Fusion flow cytometer.  Single cell suspensions were prepared using the 10x Genomics Single Cell 3' v2 Reagent Kit user guide. Approporiate volume for droplet generationas assessed by Tryptan Blue staining and hemacytometer. Reverse transcription was performed using a Veriti 96-well thermal cycler (Thermo FIsher). cDNA was recovered using 10x-associated Recovery Agent followed by amplification and purification using SPRIselect beads (Beckman) following manufacturer´s recommendation. Samples were diluted in a 4:1 ratio with elution buffer (Qiagen) prior to determining cDNA concentration using a Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared using 10x Chromium and processed as recommended by the methods provided from 10x Genomics. Raw vase call files obtained from HiSeq2500 sequencer were demultiplexed into FastQ files.