title: GSM9253842: primary spermatogonial cultures, postnatal day 7 mouse testis EVs (PND7) treated, biol rep 1; Mus musculus; RNA-Seq
library_construction_protocol: Primary spermatogonial culture was established from three postnatal day 6 male mice. Briefly, the decapsulated testes were placed in 1 mg/ml Collagenase Type IV, for 10 minutes at 37°C and tissues and cells were collected by brief centrifuge at 300g for 15s, followed by incubation in 0.25% Trypsin and 0.5 ml 5 mg/ml DNase for 10 minutes at 37°C, then stopped by the addition of 10% FBS. Tissue debris was removed by filtering through a 40-um strainer and testicular cells were collected by centrifugation at 300g for 5 min. Spermatogonia were isolated by magnetic activated cell sorting (MACS) against Thy1. 1x107 cells were resuspended in 200 μl of DMEM medium supplemented with 2% FBS and incubated with 20 μl of biotinylated Thy1.2 CD90.2 primary antibody on ice for 15 min on a slow-rocking platform. Cells were washed with PBS and resuspended with 500 μl of DMEM medium supplemented with 2% FBS before being loaded into the pre-calibrated MS column to capture Thy1+ cells. Columns were washed twice with 500 μl of DMEM medium supplemented with 2% FBS and the Thy1+ cells were eluted with 1ml of DMEM medium supplemented with 2% FBS. Then, the Thy1+ cells were cultured on the STO feeder cells in culture medium. The spermatogonial culture were treated with 1ug/ml PND7 tEVs or PBS (vehicle control) for 3 days. a.Obtain the cells of interest in suspension. b. Using a wide-bore pipette tip, gently and thoroughly mix the cells. c. Determine the cell concentration using an automated cell counter or a hemocytometer. d. Calculate the cell resuspension volume required for the desired target concentration.  To calculate cell resuspension volume, assume ~50% cell loss.  e. If significant amounts of cell clumps or debris are observed, gently mix cells by pipetting up and down 10–15x and filter cells using an appropriate cell strainer.f. Centrifuge cells at 300 rcf for 5 min at room temperature and nuclei at 500 rcf for 5–10 min at 4°C. Step 1. GEMs are generated by combining barcoded Gel Beads, a Master Mix containing  cells, and Partitioning Oil B onto  GEM-X 3' Chip.  Immediately following GEM generation, the Gel Bead is dissolved, primers are released, and any copartitioned cell is lysed. Primers containing an Illumina TruSeq Read 1 (read 1 sequencing primer, Read 1T), 16 nt 10x Barcode, 12 nt unique molecular identifier (UMI), and 30 nt poly(dT) sequence are mixed with both the cell lysate and a Master Mix containing  reverse transcription (RT) reagents. Incubation of the GEMs produces barcoded, full-length cDNA from polyadenylated mRNA.Step 2.  After incubation, GEMs are broken and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA is amplified via PCR to generate sufficient mass for library construction.Step 3.  Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. P5 and P7 adaptors, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina  amplification.