GSM2197872: purified pachytene spermatocytes RNA_n1; Mus musculus; RNA-Seq
Illumina TruSeq RNA Seq Testes from day 1 postpartum (pp), day 3 pp, day 7 pp, and adult (day 70-80 pp) were removed and decapsulated prior to RNA isolation using TRIzol (Life Technologies) from either whole tesits or enriched cells. Enrichment of specific spermatogenic cell types (leptotene/zygotene and pachytene) was achieved using the STA-PUT method based on separation by cell diameter/density at unit gravity (Bellvé, 1993). Purity of resulting fractions was determined by microscopy based on cell diameter and morphology. Pachytene cells were approximately 90% pure, with residual possible contamination from spermatocytes of slightly earlier or later developmental stages. Leptotene and zygotene cells were isolated in combination, and were >90% pure. Neither pachytene nor leptotene/zygotene fractions exhibited evidence of any contaminating somatic cells. RNA was used as the source material for mRNA sequencing and small RNA sequencing. We sequenced both small RNA and RNA from wildtype whole testis or purified meiotic cells at different stages of spermatogenesis. Sequencing libraries were genereated as replicates, from two biological replicates per sample type.