Samples were digested at 21☌ for 7.5 minutes and the reaction was stopped with EDTA. MNase digestion was completed using 200U of micrococcal nuclease in prepared digestion buffer, as per recommendations in the Brind’Amour protocol. Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. The ChIP-seq libraries for all samples were prepared using an ultra-low input native ChIP protocol, as previously described with the several modifications. Resulting libraries were pooled in equimolar quantities for 75-bp single-read sequencing on Illumina HiSeq 2000, resulting in about 15-18 million reads per sample.Ĭells for ChIP-seq were flash frozen in nuclear lysis buffer, cells for PBAT were flash frozen in PBS Subsequently, samples were subjected to NEBNext Ultra DNA library preparation for Illumina using indexed adaptors (New England Biolabs). Cells were lysed, RNA reverse transcribed and amplified according to the protocol of SMARTer Ultra Low RNA Kit for Illumina Sequencing (Version 1, Clontech). ![]() Oocytes were hand collected and washed in M2 medium (Sigma), followed by 2 consecutive washes in PBS.įor single oocyte RNA profiling, four GV oocytes from Mll2 KO and WT mice were collected for each genotype. Ovaries were collected from 5, 10, 15, and 25 day-old C57BL/6Babr or 21 or 25-day-old Mll2Gdf9cKO or 25-day-old Dnmt3a/bZp3cDKO mice and digested using 2mg/mL collagenase and 0.02% trypsin solution, gently agitating at 37☌ for 20-30 minutes. Time-point in reprogramming: postnatal day 21 GEO help: Mouse over screen elements for information.
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