Illumina NovaSeq 6000 paired end sequencing; RNA-seq in wild type and Bmi1 knockdown undifferentiated spermatogonia
RNA-seq in wild type and Bmi1 knockdown undifferentiated spermatogonia
Isolation and culture of undifferentiated spermatogonia followed the protocols with little modifications. Briefly, testis was cut off from 6 day-old pups and dissociated by a two-step enzymatic digestion. About 20 mice were used to isolate wild undifferentiated cells, and about 40 mice were used to isolate undifferentiated cells then knocked down Bmi1 by siRNA. Cells were resuspended in IMDM/FBS medium and plated onto dishes coated by 0.2% (w/v) gelatin. An hour later, the suspended cells were transferred to a new dish coated by gelatin. This step was repeated twice, then incubated overnight. Somatic cells were adhered in dishes, undifferentiated spermatogonia were loosely attached. 4x10^6 cells were used in wild and Bmi1 knockdown group. Total RNA was isolated by Trizol (Invitrogen) according to the manufacturerâ€™s protocols. mRNA was purified by NEBNext Poly(A) mRNA Magnetic Isolation Beads (NEB, USA). The RNA libraries were constructed using the NEBNext Ultra Directional RNA Library PrepKit (NEB, USA).