Illumina HiSeq 2500 sequencing; Transcriptional start site mapping of P14 WT and P14 Tbpl2-/- mutant mouse oocytes
Transcriptional start site mapping of P14 WT and P14 Tbpl2-/- mutant mouse oocytes
After dissection, ovaries are freed from adhering tissues in 1x PBS. Series of 6 ovaries were digested in 500 μL of 2 mg/mL Collagenase (SIGMA), 0.025% Trypsin (SIGMA) and 0.5 mg/mL type IV-S hyaluronidase (SIGMA), on a thermomixer at 600 rpm for 20 minutes. The digestion was then stopped by the addition of 1 mL of 37°C pre-warmed αMEM - 5% FBS. The oocytes were then size-selected under a binocular. Pool of 100-200 oocytes collected were washed through several M2 drops, and total RNA was isolated using NucleoSpin RNAXS kit (Macherey-Nagel) according to the user manual. RNA quality and quantity were evaluated using a Bioanalyzer. Between 5-10 ng of RNA was obtained from each pool of oocytes. Twenty-eight and 13 ng of total RNA isolated from P14 oocytes (biological replicate 1 and replicate 2, approximately 500-1000 oocytes pooled for each replicate) and 15 ng of total RNA isolated from P14 Tbpl2-/- mutant oocytes (approximately 550 pooled oocytes) were used for SLIC-CAGE TSS mapping. Briefly, 5 μg of the carrier RNA mix were added to each sample prior to reverse transcription, followed by the cap-trapping steps designed to isolate capped RNA polymerase II transcripts. The carrier was degraded from the final library prior to sequencing using homing endonucleases. The target library derived from the oocyte RNA polymerase II transcripts was PCR-amplified (15 cycles for P14 WT, 16 cycles for P14 Tbpl2-/- mutant) and purified using AMPure beads (Beckman Coulter) to remove short PCR artifacts (< 200bp, size selection using 0.8 x AMPure beads to sample ratio)