Illumina NovaSeq 6000 paired end sequencing; ChIP-seq in wild type and Bmi1 knockdown undifferentiated spermatogonia
ChIP-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 for each ChIP-seq experiment. Cells were cross-linked using a final concentration of 1% formaldehyde (Thermo Fisher, USA) at room temperature for 10 min, then stopped by a final concentration of 125mM Glycine (Sangon, China) at room temperature for 5min, subsequently washed twice with PBS. Cells were isolated nucleus according to previously described. Chromatin was sheared by Covaris sonicator (Cavaris, USA). The fragmented chromatin was immunoprecipitated with Magna ChIPTM Protein A+G Magnetic beads (Millipore, USA) coupled with antibodies overnight at 4°C. Then, the beads were washed with low salt, high salt, LiCl and TE buffer. Bound chromatin was eluted and reverse crosslinked.  ChIP and input DNA were constructed libraries using KAPA Hyper Prep Kit (KAPA, USA) according to the manufacturer's instructions