Hiseqv4

ChIP was performed as previously described (Lee et al., 2006 (link)). In brief, 150 μl of ovaries were dissected into ice-cold PBS, crosslinked with 1.8% formaldehyde in PBS for 10 min at room temperature, quenched with Glycine, rinsed in PBS and flash frozen in liquid nitrogen after removing all PBS. Frozen ovaries were disrupted in PBS using a dounce homogenizer, centrifuged at low speed and the pellet was resuspended in lysis buffer. For ChIP from OSCs 5–10 million cells were crosslinked, quenched, and lysed. Sonication (Bioruptor) resulted in DNA fragment sizes of 200–800 bp. Immunoprecipitation with specific antibodies was done overnight at 4 °C in 350–700 μl total volume using 1/3 to 1/4 of chromatin per ChIP (antibodies are listed in Key Resource Table). Then, 40 μl Dynabeads (equal mixture of Protein G and A, Invitrogen) were added and incubated for 1 hr at 4°. After multiple washes, immuno-precipitated protein-DNA complexes were eluted with 1% SDS, treated with RNAse-A, decrosslinked overnight at 65 °C, and proteins were digested with proteinase K before clean-up using ChIP DNA Clean & Concentrator columns (Zymo Research). Barcoded libraries were prepared according to manufacturer’s instructions using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB), and sequenced on a HiSeqV4, NextSeq550, or NovaSeqSP (Illumina).

Total RNA was extracted in triplicates from approximately 5 × 10⁶ TCs per cell line at about 80 percent confluence in triplets using the RNA extraction kit from Qiagen (RNeasy Mini Kit, #74104). The cDNA was synthesized using 500-ng total RNA with the cDNA synthesis kit from Quantseq (#015, Lexogen, Vienna, AT). RNA sequencing was conducted on Illumina HiSeqV4 instrument using single-end read mode (50-nt read length) on HiSeq5A (Illumina, San Diego, CA). For normalization, counts per million mapped fragments (FPM/CPM) using column sums of the raw counts were applied. Average values of the triplicates were used for analysis.