title: GSM8213270: 613DNA1; Mus musculus; Bisulfite-Seq
library_construction_protocol: DNA was isolated from tissue punches using Qiagen QIAamp DNA Micro Kits (Qiagen, Cat. # 56304) according to manufacturer instructions for Isolation of Genomic DNA from Tissues with the following modifications and optional steps included to increase yield. Prior to addition of ATL buffer to punches, 80 µL PBS was added and a 1.5 mL tube pestle was used to manually homogenize the sample. 100 µL ATL buffer was added to each sample. For the proteinase K digestion step, samples were incubated at 56°C overnight. Carrier RNA was added to Buffer AL. The incubation time after addition of 100% EtOH was increased to 10 minutes. The incubation time for the elution step was increased to 5 minutes and this step was repeated a second time. DNA was eluted in 54 µL of 10 mM Tris-HCl, pH 8.0. SeqCap Epi custom bait probes for regions of interest were designed using the Roche NimbleDesign software.Capture hybridization-sequencing libraries were prepared by the Van Andel Institute Genomics Core from 100 ng of high molecular weight DNA using the Accel-NGS Methyl-Seq DNA Library kit (v3.0) (Swift Biosciences, Cat. #30024). DNA was sheared following manufacturer's protocol to an average size of 250bp, and sheared DNA was bisulfite converted using the EZ DNA Methylation-Gold kit (Zymo Research, Cat. #D5005) with an elution volume of 15 µL. Following adapter ligation, 8 cycles of library amplification were performed. Amplified libraries were pooled in batches of 8 and targeted enrichment of our custom regions was performed using Roche SeqCap Epi developer probes (Supplementary File 1) and SeqCap HyperEpi workflow starting at step 4.0 with the following modifications. The capture was performed using IDT xGen Universal blockers to replace the SeqCap HE Universal Oligo and SeqCap HE Index Oligo. The post-capture amplification was also adjusted to 11 cycles of amplification and the final extension changed from 30 seconds to 1 minute. Quality and quantity of the finished library pools were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.) and QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA. Sequencing (100 bp, paired end) was performed on an Illumina NovaSeq6000 sequencer using an S4, 200 bp sequencing kit (Illumina Inc., San Diego, CA, USA) with 10% PhiX included to improve base diversity. Base calling was done by Illumina Real Time Analysis 3 and output of NextSeq Control Software was demultiplexed and converted to FastQ format with the Illumina Bcl2fastq software (version 1.9.0). Capture hybridization sequencing (targeted bisulfite-seq)