Illumina HiSeq 2000 paired end sequencing; Defective germline reprogramming rewires the spermatogonial transcriptome
Defective germline reprogramming rewires the spermatogonial transcriptome
Isolated postnatal day 14 testes from mice heterozygous for the Miwi2- tdTomato transcriptional allele were dealbulginated and digested with collagenase (0,5 mg/ml, Sigma) at 32oC for 10 minutes shaking in 1ml of DMEM media (Life technologies) supplemented with Pen/Strep (Life technologies), NEAA (Life technologies), sodium pyruvate (Life technologies) and sodium lactate (Sigma). After digestion seminiferous tubules were collected by centrifugation at 1000 rpm for 5 min. Single cell suspension was prepared by further digesting seminiferous tubules in 1 ml of 0,05% trypsin (Life technologies) and DNase (Sigma), added to 0,05 mg/ml of final concentration, at 32oC for 8-10 minutes shaking. After digestion trypsin was neutralized by adding 100 μl of FCS. Cells were spun and resuspended in PBS with 3% FCS. Germ cells were stained with following antibodies: c-Kit 1:800 (eBioscience), CD45-biotin 1:400 (eBioscience), CD51-biotin 1:100 (Biolegend), streptavidin-qDot 1:50 (eBioscience), CD9 1:50 (eBioscience) in PBS with 3% of FCS and 0,01% sodium azide. SYTOX Blue (Life technologies) was used as a living dye. Target population was selected by excluding cell doublets and CD45pos and CD51pos cells by initially choosing to analyze cells with low side scatter and negative for live cell dye SYTOX Blue. Among CD45neg and CD51neg cells, a target population was identified as c-Kitneg and having a live fluorescence of tdTomato and being CD9pos. Cells were sorted with an 85μm nozzle using a FACSAria II SORP (BD Biosciences). This strategy results in the isolation of undifferentiated spermatogonia with a purity of greater than 98%. Sorted cells were digested with proteinase K at 55C over night. The DNA was extracted using 1 Volume Phenol/Chloroform twice followed by the addition of 1 Volume chloroform and then DNA was precipitated by addition of 1 volume isopropanol with the addition of linear acrylamide and washed with 70% Ethanol before resuspension. For bisulfite conversion DNA was isolated from sorted cells as described above. Whole genome bisulfite sequencing libraries were generated using a post-bisulfite adaptor tagging (PBAT) method as previously described4 using 10 cycles of PCR amplification. Three biological replicates were generated per genotype.
Experimental Factor: replicate
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