GSM3402315: sHet-mPN-RRBS.rep2; Mus musculus; Bisulfite-Seq
Genomic DNA was isolated by conventional phenol extraction and proteinase K treatment. For sperm samples, 40 mM DTT were added during cell lysis. RRBS for oocyte and PGC samples: For oocytes, before MspI digestion, 5 µl of lysis buffer (20 mM Tris-EDTA [pH 8.0], 20 mM KCl, and 0.3% Triton X-100, 1 mg/ml QIAGEN Protease) was directly added to the frozen samples (in < 1µl PBS) followed by 3 hours of incubation at 50 ℃ and 30 minutes of inactivation at 75 ℃. Then to the same tube, 13 µl of MspI digestion master mix containing 10 units of MspI (Thermo Scientific) and 1.8 µl 10x Buffer Tango (Thermo Scientific) and 0.5pg unmethylated λ DNA (Thermo Scientific) was added followed by 3 hours of incubation at 37 ℃. For PGCs, the purified genomic DNA was directly digested with MspI in an 18 µl total volume. The digested DNA was then filled-in and tailed with an extra A to the 3' end by adding 2 µl of end-preparation master mix containing 5 units of Klenow Fragment (exo-; Thermo Scientific), 0.2 µl of 10x Buffer Tango, 0.4 mM dATP, and 0.04 mM each of dGTP and dCTP (Thermo Scientific), followed by 20 minutes of incubation at 30 ℃, 20 min of incubation at 37 ℃ and 15 minutes of inactivation at 80 ℃. Adaptor ligation was then performed by adding 5 µl of ligation master mix, which contains 30 Weiss Units of T4 DNA Ligase (HC, Thermo Scientific), 0.5 µl of 10x Buffer Tango, 5 mM ATP (Thermo Scientific), and 150 nM of methylated custom adaptor (Forward: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3', Reverse: 5'-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3', where -s- indicates phosphorothioate bond). After three incubation steps (16 ℃ for 30 min, 4 ℃ overnight, and 65 ℃ for 20 min), the reaction mix was subjected to EpiTect Bisulfite Conversion Kit (QIAGEN) with modified program: 95 C for 5 min, 60 C for 25 min, 95C for 5 min, 60C for 85 min, 95C for 5 min, 60C for 175 min, 3x (95 C for 5 min, 60 C for 90 min). After clean up, the optimal, minimum PCR cycle number required to generate the final libraries was determined using diagnostic PCRs. Remaining bisulfite converted products were PCR amplified with NEBNext Multiplex Oligos (NEB) and KAPA HiFi Hotstart Uracil+ ReadyMix (Kapa Biosystems). Final libraries were obtained by size selection of 150-500 bp DNA fragments using AMPure XP beads (Beckman Coulter). RRBS for sperm: 100ng genomic DNA was digested with 10 units of MspI in a 18 µl total volume at 37 ℃ overnight. The digested DNA was then filled-in and tailed with an extra A to the 3' end by adding 2 µl of end-preparation master mix containing 5 units of Klenow Fragment exo- , 0.2 µl of 10x Buffer Tango, 5 mM dATP, and 0.5 mM each of dGTP and dCTP, followed by 20 minutes of incubation at 30 ℃, 20min of incubation at 37 ℃ and 15 minutes of inactivation at 80 ℃. Adaptor ligation was then performed at 20 ℃ for 15min using NEBNext Ultra Ligation Module (NEB) by adding 15 µl of ligase master mix, 1 µl of ligation enhancer, 2.5 µl of 1.5 µM methylated custom adaptor and ddH2O to a total volume of 80 µl. The reaction mix was then size selected of 150-500 bp DNA fragments using AMPure XP beads. The size-selected sample was then subjected to EpiTect Bisulfite Conversion Kit (QIAGEN) with modified program: 95 C for 5 min, 60 C for 25 min, 95C for 5 min, 60C for 85 min, 95C for 5 min, 60C for 175 min, 3x (95 C for 5 min, 60 C for 90 min). After clean up, the optimal, minimum PCR cycle number required to generate the final libraries was determined using diagnostic PCRs. Remaining bisulfite converted products were PCR amplified with NEBNext Multiplex Oligos (NEB) and KAPA HiFi Hotstart Uracil+ ReadyMix (Kapa Biosystems). Final libraries were obtained by AMPure XP beads clean-up of the PCR product. RNA-seq for oocyte samples: cDNA libraries were synthesized and amplified using SMARTer Ultra Low Input RNA Kit for Sequencing (Clontech) following manufacturer's instructions. The purified cDNA was fragmented with Covaris M220 sonicator, and then subjected to Illumina sequencing library construction with KAPA Hyper Prep kits (Kapa Biosystems) following manufacturer's instructions.
GEO Accession
GSM3402315