GSM8013369: KO1_BS; Mus musculus; Bisulfite-Seq
The small intestine was dissected from E18.5 embryos, opened longitudinally, and placed on ice-cold PBS containing 30mM EDTA and 1.5mM DTT for 20 min on ice. Next, the intestine was transferred to a tube containing 30mM EDTA in PBS, incubated at 37ºC for 10min and, then, vigorously shaken for 30s to release the intestinal epithelium from the lamina propia. After removing the intact intestinal muscle, the villi suspension was used for 5hmC genome wide profiling. Uniquely indexed libraries were generated using the Ovation Ultralow Methyl-Seq DR Multiplex with TrueMethyl oxBS kit and workflow (Tecan). Each library was split into two aliquots for chemistry as follows: 2/3 were oxidized and bisulfite converted (oxBS), the other 1/3 was subjected to a mock oxidation before bisulfite conversion. 2microl of the resulting material was assessed by qPCR to determine the optimal number of PCR amplification cycles. After amplification the libraries were normalized to 2nM and pooled according to the desired plexity, clustered at 6.5pM on single read flow cell and sequenced for 100 cycles on an Illumina NovaSeq. Library preparation for RRoxBS libraries, sequencing, post-processing of the raw data, data alignment and methylation calls were generated at the Epigenomics Core at Weill Cornell Medicine (New York, USA).