RNA Isolation and Sequencing Protocol

Isolation and library generation was done as previously described (52 (link)). Briefly, RNA was extracted using the standard protocol within the AllPrep DNA/RNA Micro kit (Qiagen, Germantown, MD, USA). RNA quality from sorted nuclear RNA was determined based on Bioanalyzer Picochip analysis (Agilent, Santa Clara, CA, USA). Isolated RNA was amplified, made into cDNA, sheared, and libraries were made. The library was quantified using the Qubit dsDNA kit (Invitrogen) and Kapa library quantification kit (KapaBiosystems, Boston, MA). cDNA libraries were pooled, clustered on the cBot and subject to 100 or 125 base pairs paired end reads on the HiSeq 2000 or 2500 (Illumina, San Diego, CA, USA).