Cytoplasmic and nuclear RNA purification and sequencing

We used the Norgen Biotek Corp. Cytoplasmic and Nuclear RNA Purification Kit (21000, 37400) following the manufacturer's protocol including DNase I treatment, lysing 15 mg of tissue using Lysis Buffer J, and isolating fractions by centrifugation before extracting RNA using spin column chromatography.

RNA-sequencing libraries were prepared using poly(A)-selection (“poly(A)”; Illumina TruSeq Stranded Total RNA Library Prep Kit, RS-122-2201) and rRNA depletion (“Ribo-Zero”; Illumina Ribo-Zero Gold Kit [Human/Mouse/Rat] MRZG126) protocols. Libraries were sequenced on one Illumina HiSeq 2000 lane. Illumina Real Time Analysis (RTA) module performed image analysis and base calling, running the BCL converter (CASAVA v1.8.2), generating FASTQ files containing the sequencing reads. One adult nuclear Ribo-Zero sample failed quality control and was discarded. “Br5339C1_polyA” and “Br5340C1_polyA” FASTQ files were downsampled to 24 million total reads by concatenating FASTQ files across sequencer lanes for each sample, keeping read 1 and read 2 files separate, using the shuf unix command to shuffle the reads then printing the top 24 million shuffled records to new FASTQ files.