RNA-seq of mouse spermatogonia cells at three developmental stages (PND8, PND15 and adult)
RNA-seq of mouse spermatogonia cells at three developmental stages (PND8, PND15 and adult)
For pup testis, dissociated cells were stained with BV421-conjugated anti-β2M, biotin-conjugated anti-THY1 (53-2.1), and PE-conjugated anti-αv-integrin (RMV-7) antibodies. THY1 was detected by staining with Alexa Fluor 488-Sav. For adult testes, cells were stained with anti-α6-integrin (CD49f; GoH3), BV421-conjugated anti-β2 microglobulin (β2M; S19.8), and R-phycoerythrin (PE)-conjugated anti-THY1 (CD90.2; 30H-12) antibodies. α6-Integrin was detected by Alexa Fluor 488-SAv after staining with biotin-conjugated rat anti-mouse IgG1/2a (G28-5) antibody. Prior to FACS, 1 μg/ml propidium iodide (Sigma) was added to the cell suspensions to discriminate dead cells. All antibody incubations were performed in PBS-S for at least 30 min at 4oC followed by washing in PBS-S. Antibodies were obtained from BD Biosciences (San Jose, United States) unless otherwise stated. Cell sorting was performed at 4oC on a FACS Aria III 5L using an 85μm nozzle at the Cytometry Facility of University of Zurich. RNA sequencing libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v3 (Takara, Cat# 634486) following the recommended protocol with minor adjustments. Adult spermatogonial cells libraries were prepared using the option to start with 1000 cells. Cells were sheared for 6 minutes at 85 °C. After reverse transcription, samples were barcoded using the SMARTer Unique Dual Index Kit (Takara, Cat# 634452). Five PCR cycles were performed and the PCR products were purified using AMPure XP reagent (Beckman Coulter Life Sciences, Cat# A63881; bead:sample ratio 0.8X). Ribosomal cDNA was depleted following the protocol and final library amplification was performed with twelve PCR cycles. Libraries were purified twice using AMPure XP reagent (bead:sample ratio 1X) and eluted in Tris buffer.Concentration and fragment length distribution of all samples were measured using Qubit 2.0 (Invitrogen) and Bioanalyzer (Agilent). Samples were pooled at the equal molarity, as determined in a 100 – 800 bp window on the Bioanalyzer.