title: GSM9294228: scRNA-seq_3-week Vehicle-Recovery Replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: One testis was collected for single cell RNA sequencing (n = 3 mice). Testes were detunicated and minced on ice and transferred to a 50 mL tube containing 40 ml of collagenase IV (1 mg/mL, Sigma-Aldrich, C4-BIOC) and DNase I (7 mg/mL, Sigma Aldrich DN25) in Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11-965-092). Tissue was incubated in a 37 °C incubator for 15 minutes. Tubule fragments were then centrifuged at 60 x g for 10 minutes, the supernatant discarded, washed with 15 mL Dulbecco's Phosphate Buffered Saline (dPBS, Sigma-Aldrich, D8537), and centrifuged again at 60 x g for 10 minutes. The fragments were then incubated in 10 mL 0.05% trypsin-EDTA (Invitrogen, 25200056) solution in 37 °C incubator for 12 minutes. Trypsin was quenched with 10% v/v fetal bovine serum (FBS, Thermo Fisher Scientific, A5256701), cells were passed through a 40 µm filter, spun down at 300 x g for 5 minutes, and resuspended in 1mL 0.04% bovine serum albumin (BSA, Sigma-Aldrich, A4737) in 1X dPBS. Library was performed according to the manufacturer's instructions (single cell 3' v3.1 protocol 10x genomics). Briefly, testicular cells were resuspended in the master mix and loaded together with partitioning oil andn gel beads into the chip to generate a gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which containes an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane Dynabeads, amplified by PCR and the apropiated size fragments were selected with SPRIselect reagent for subseqeunt library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.