RNA Isolation and Sequencing Protocol

RNA isolation was performed as for PHDFs and MEFs. Cells were lysed with 27-gauge, 1/2-in. needles and then homogenized with QIAshredder columns (Qiagen). Total RNA from triplicate experiments was purified with the RNeasy Plus kit (Qiagen). The quality of purified total RNA samples was determined with an Agilent 2100 Bioanalyzer, and only samples with an RNA integrity number (RIN) of 9 or higher were used. RNA concentration was measured with a Qubit fluorimeter prior to library prep. Four micrograms of total DNase-treated RNA was run through the TruSeq Stranded Total RNA LT sample prep kit from Illumina as previously described (18 (link)). Samples were quantified by Qubit before being normalized, pooled, and then sequenced on the Illumina HiSeq 2500 sequencer with SBS v3 reagents. Each sample was sequenced at a depth of at least 25 million 50-nucleotide single-end reads.