GSM8941311: OCorg3P_concat; Mus musculus; Bisulfite-Seq
Cell pools from all organoids were lysed in 2% SDS and 25 mM EDTA, overnight. Ammonium acetate (10 M) was added to precipitate SDS with proteins, which were removed by centrifugation at 12,000 rpm. DNA was then precipitated with isopropanol, washed in 70% ethanol, and dissolved in LTET (10 mM Tris, 0.1 mM EDTA, 0.1% Tween 20). DNA concentration was measured using the Qubit dsDNA Broad Range kit (Thermo Fisher, #Q32850). Two micrograms of DNA were digested with 100 U of MspI endonuclease (NEB, #R0106), at 37°C, for 3.5 h. The Klenow fragment (3′ > 5′ exo-  ) DNA polymerase (NEB, #M0212) was used to fill in 3′ recesses and generate 3′ dA overhangs. Size selection was performed using AMPure XP beads (Beckman, #A63881), with a 2X bead-to-DNA volume ratio. Methylated sequencing adapters (NEB, #E7535) were ligated to DNA fragments with T4 DNA ligase (NEB #M0202), at 16°C overnight. Adapters were cleaved by incubation with USER enzyme (NEB #M5505), at 37°C, for 15 min. Dual size selection with AMPure XP beads removed DNA fragments greater than 500 bp and less than 150 bp. Purified DNA fragments were bisulfite-converted using the EpiTect Bisulfite Conversion Kit (Qiagen, #59104). The bisulfite-converted fragments were cleaned using a 3X AMPure bead-to-DNA ratio. NEBNext Index i7 primers (NEB #7335, #7500) were used to amplify libraries with EpiMark Hot Start Taq (NEB, #M0490), for 15 cycles. Library concentrations were measured using the Qubit dsDNA High Sensitivity kit (Thermo Fisher, #Q32851). The Agilent Bioanalyzer determined size distribution and molarity using the Agilent High Sensitivity DNA kit (Agilent, #5067-4626). Libraries were pooled to a 10 nM concentration and spiked with 25% phiX (Illumina, #FC1103001) for sequence diversity. Sequencing occurred over several years at multiple facilities.