GSM5243399: Z6_Zfp541-KO_lP_360h_rep2; Mus musculus; RNA-Seq
Testicular tissues were disassociated enzymatically using enzyme mixtures comprising of 1 mg/mL collagenase IV (GIBCO,17104-019), 1.5 mg/mL hyaluronidase (SIGMA，H2126), 1 mg/mL trypsin (SIGMA，T1426), and 1 mg/mL DNase I (SIGMA, DN25), dissolved in DMEM medium at 35℃ with gentle agitation for 20 mins. Subsequently, the cell suspension was filtered through a 40 μm cell strainer, centrifuged at 250×g for 5 min at 4 °C, the supernatant was removed and cells in the pellet were resuspended in cold PBS (Phosphate buffer saline) containing 0.05% BSA carefully. Single-cell RNA-seq by Smart-seq2 were performed as previously reported (Chen et al., 2018b; Li et al., 2017; Picelli et al., 2013). Briefly, the single spermatogenic cells were picked into lysis buffer containing reverse transcription primers by mouth pipette. After the first-strand synthesis, the second-strand cDNAs were synthesized, and the cDNAs were amplified by 17 cycles of PCR. Samples with different barcodes were pooled together. Biotinylated pre-indexed primers were used to amplify the PCR product by an additional 4 cycles of PCR to introduce biotin tags to the 3′ ends of the amplified cDNA. cDNA was sheared to approximately 300 bp by Covaris S2, and the 3′ terminal of the cDNA was captured by Dynabeads® MyOne Streptavidin C1 beads (Thermo Fisher). The RNA-seq library was constructed using a Kapa Hyper Prep Kit (Kapa Biosystems) and subjected to 150 bp paired-end sequencing on an Illumina HiSeq 4000 platform (sequenced by Novogene).
GEO Accession
GSM5243399