title: GSM7135565: TDP-43 cKO scRNAseq rep1; Mus musculus; RNA-Seq
library_construction_protocol: PND 24 (post-natal day 24) mice of TDP-43 cKO (N=3), Nelf-B cKO (N=2) and corresponding littermate/ non-littermate control (N=4) were used for single-cell RNA-seq. Testes were dissected and collected in 35 mm plate with ice cold DMEM (DMEM henceforth). Testes were decapsulated and the seminiferous tubules were minced thoroughly with micro squeeze scissors. The resulting minced tubules were collected in a 15 ml conical centrifuge tube with 10 ml DMEM and washed twice at 0.1 xg for 1 min in an Eppendorf swing bucket centrifuge (model 5702 R). After the final wash the pellet was suspended in 1 ml DMEM and digested with 250 µl of 1% collagenase and 10 µl of 1% DNase at 35.5°C for 20 min in a rotating shaker (100 rpm) until tubules were well separated. The enzymatic reactions were stopped by diluting the enzymes with excess amount of (12 ml) DMEM and washed twice at 0.1 xg for 1 min. After the final wash the pellet was suspended in 1 ml DMEM and put through a second enzymatic digestion with 1 mL of 0.25% trypsin, 350 µl of 1% hyaluronidase, 200 µl of 1% collagenase, and 10 µl of 1% DNase, at 35.5°C for 20 min in the rotating shaker. The enzymatic digestions were stopped by adding 12 ml of DMEM. The resulting single cell suspensions were filtered through 40 μm EZFlow nylon mesh (Foxx Life Sciences, Londonderry, NH) and the filtered samples were centrifuged at 0.3 xg for 5 min. The supernatant was discarded carefully, and single cells pellet was suspended in DMEM with 1-2% BSA. Finally, the cell suspensions were passed through Flowmi 40 μm cell strainer (SP Bel-Art, Wayne, NJ) to discard clumped cells and collected in 1.5 ml microfuge tubes. The tubes were stored on ice and proceeded for the 10X genomic facilities. Single-cell 3' cDNA libraries were prepared at the DNA Services laboratory of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. Single-cell suspensions were delivered to the facility and were counted and checked for viability >80% using the Nexcelom K2 brightfield/dual florescence cell counter (Nexcelom Biosciences, Lawrence MA) with AO/PI staining, then washed with PBS buffer containing 2.0% BSA, then recounted for library preparation. The target number of cells (5,000) from each population were converted into individually barcoded cDNA libraries with the Single-Cell 3' NextGEM v3.1 Chromium kit from 10X Genomics (Pleasanton, CA) following the manufacturer's protocols. Following ds-cDNA synthesis, individually-barcoded dual-index libraries compatible with the Illumina chemistry were constructed. The final libraries were quantitated on Qubit (Life Technologies, Grand Island, NY) and the average size determined on the AATI Fragment Analyzer (Agilent Technologies, Santa Clara, CA).  Libraries were pooled evenly and the final pool diluted to 5nM final concentration. The 5nM dilution was further quantitated by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA).