Allprep dna rna plus micro kit

Genomic DNA from sorted HSPCs was isolated using the AllPrep DNA/RNA Plus micro kit (QIAGEN). Approximately 200 ng of genomic DNA was sonicated followed by end-repair, A-tailing, and adaptor ligation using standard Illumina protocols. Ten percent of the volume was retained for Input. Immunoprecipitation with anti-5-hmC antibodies (1 μg/mL, Active Motif) was performed at 4°C overnight and immunoprecipitated DNA was recovered using protein G magnetic beads (Invitrogen). After proteinase K digestion, DNA was purified from all input and immunoprecipitated samples and amplified by PCR for library preparation. Libraries were sequenced by an Illumina HiSeq 2500 sequencer by the Cancer Genomic Core Facility at University of Miami. Reads were trimmed using cutadapt (–m25), aligned using bowtie2 (v2.0.5) to the mouse mm10 genome build, quality filtered (mapq > 30), and deduplicated. Peaks were called using macs2 (-g mm --nomodel --extsize 200 –B) and differential peaks were determined using macs2 bdgdiff from the intermediate lambda and pileup bed graphs generated from macs2 call peaks.