title: GSM2628494: Epi_WT_7_Perturbation-RRBS; Mus musculus; Bisulfite-Seq
library_construction_protocol: Genomic DNA and mRNA purifications from low input samples were performed as described by Maculay et al. 2015 with modifications. Briefly, the cells were mixed with 15 µl of RLT plus buffer (QIAGen) containing 1 U/µl of RNase inhibitor (SUPERase·In, ThermoFisher Scientific), 1% ß-mercaptoethanol (Sigma-Aldrich), and were then transferred to one well in a 96-well DNA LoBind plate (Eppendorf). After adding 10 µl of M-280 streptavidin bead-conjugated RT primer to each sample, the reaction was incubated at 72˚C for 3 min in a thermocycler followed by incubation at room temperature for 25 min with gentle rotation. The genomic DNA and mRNA were separated in a DynaMag™-96 Side Magnet (ThermoFisher Scientific). Bead-tagged mRNA was subjected to reverse transcription as described previously in Maculay et. al 2015 and the genomic DNA in the supernatant was transferred to a fresh 96-well DNA LoBind plate. After reverse transcription, the cDNA was PCR amplified and RNA-seq library was generated according to the Smart-seq 2 protocol. Indexed RNA-seq libraries were pooled and sequenced in an Illumina Hiseq2500 sequencer.  Genomic DNA was isolated utilizing 1x Agencourt AMPure beads (Beckman Coulter) and was eluted to 15 µl of low TE buffer. RRBS library was generated as reported previously in Gu et. al 2013 with modifications. We utilized the CutSmart buffer (New England Biolabs) for all three enzymatic reactions including MspI digestion, end-repair/A-tailing and T4 DNA ligation. To minimize DNA loss, DNA purification step was eliminated after each enzymatic reaction. Briefly, the genomic DNA was digested by 16 units of MspI (New England Biolabs) for 80 min at 37˚C, and followed by heat inactivation at 65˚C for 15 min. The digested DNA fragments were end-repaired and A-tailed by adding 4 units of Klenow exo- (New England Biolabs), 0.03 mM dCTP, 0.03 mM dGTP and 0.3 mM dATP; ant the reaction was carried out at 30˚C for 25 min; 37˚C for 25 min followed by incubation at 70 ˚C for 10 min to inactive the enzyme. We then ligated the A-tailed DNA fragments with indexed adapters overnight at 16 ˚C by adding 2,000 units of T4 DNA ligase and 0.75 mM ATP and 7 nM of the adapters. The T4 ligase was heat-inactivated at 65 ˚C for 15 min before pooling libraries together. To remove adapter dimers, the library pool was cleaned up using 1.8X AMPure beads and the adapter-tagged DNA fragments were eluted to 30 µl of low TE buffer. The bisulfite conversion of the adapter-tagged DNA fragments ware conducted using a QIAGen EpiTect Fast Bisulfite Conversion Kit following the manufacturer’s instructions with a minor modification. We extended the bisulfite conversion time from 2 cycles of 10 min to 2 cycles of 20 min to achieve bisulfite conversion rates >99%. The bisulfite converted DNA fragments were PCR amplified according to the following thermocycler settings: 98 ˚C for 45 s, 6 cycles of 98 ˚C for 20 s, 58 ˚C for 30 s, 72 ˚C for 1 min and then 8-10 cycles of 98 ˚C for 20 s, 65 ˚C for 30 s, 72 ˚C for 1 min followed by a final extension cycle of 5 min at 72 ˚C. The PCR amplified library DNA was cleaned up using 1.3X AMPure beads and the RRBS libraries were paired-end sequenced for 2x100 cycles.  Only instances where the matched pool of Epiblast and ExE from a given replicate both had over 1 million CpGs covered at ≥5x were included for downstream analysis.  For each sample, 10 µl of M-280 streptavidin beads (ThermoFisher Scientific) were prepared per the manufacturer’s recommendations. Specifically, after washing with Solution A (0.1 N NaOH, 0.05 M NaCl) and B (0.1 M NaCl) sequentially, the beads were resuspended in 10 µl of 2x Washing and Binding buffer (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl) and then mixed with an equal volume of 2 µM of RT primer (Maculay et al. 2015). The mixture was incubated for 15 min at room temperature with gentle rotation. The bead-bound RT primer was collected in a magnate and was subsequently resuspended in 10 µl of binding buffer (10 mM Tris-HCl (PH 8.0), 167 mM NaCl, 0.05% Tween-20).
attributes.tag: GEO Accession
attributes.value: GSM2628494