Rat kangaroo paired-end PtK2 transcriptome
Rat kangaroo PtK2 cell RNA extraction: PtK2 cells were plated at 30% confluency on a 10 cm tissue culture dish and grown unsynchronized at 37.0°C and 5% CO2 for 48 h.  RNA was extracted and purified using the RNeasy® Midi Kit (Qiagen #75142).  Cell culture media was aspirated out of the dish and 1 mL of Buffer RLT (with β-ME) was added to lyse cells.  Lysate was collected using cell lifters (Corning), vortexed for ~10 sec, and passed through a 27-gauge needle 10 times to homogenize cells. The purification continued according to the manufacturer’s protocol.    Rat kangaroo cDNA library construction and sequencing: Total RNA quality was assessed by spectrophotometer (NanoDrop, Thermo Fisher Scientific Inc.) and the Agilent 2100 Bioanalyzer (Agilent Technologies). mRNA was purified from 1 μg of total RNA using poly(A) tail selection.  RNA sequencing libraries were generated using the TruSeq RNA Sample Prep Kit (Illumina) with multiplexing primers, according to the manufacturer’s protocol.  The cDNA library was constructed with average inserts of 275 bp, with non-stranded library preparation.  Fragment size distributions were assessed using the Bioanalyzer 2100 and the DNA high-sensitivity chip.  Library concentrations were measured using the KAPA Library Quantification Kit (Kapa Biosystems Inc.) and run on the Illumina HiSeq2500.  Sequencing was performed via a paired-end 150 cycle rapid run on 2 lanes of the Illumina HiSeq2500, generating more than 300 million pairs of reads as desired.