Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: For all listed samples, we isolated total RNA using a Qiagen Rneasy Plus Mini™ Kit (Qiagen®, Valencia, CA; cat. no. 74134). We constructed cDNA libraries using two Illumina® TruSeq™ Stranded mRNA Library Preparation Kits (Illumina®, cat. no. RS-122-2101 and RS-122-2102, Adaptor Sets A and B) (Figure 1 ). Libraries were prepared in two sets of 24 and randomized to account for batch effects. Briefly, we removed ribosomal and other non‑coding RNAs to purify messenger RNA (mRNA) and fragmented the isolated mRNA. We synthesized first strand cDNA using SuperScript III Reverse Transcriptase (Invitrogen™, cat. no. 18080-044), and second strand cDNA using the 2'-deoxyuridine 5'-triphosphate (dUTP) nucleotide method for strand specificity. We then ligated a unique adapter index to each cDNA library and amplified the libraries through 15 cycles of polymerase chain reaction (PCR). cDNA purification steps were completed with PCRClean™ DX paramagnetic beads (Aline Biosciences®, cat. no. C-1003-5) and Agencourt AMPure™ XP beads (Beckman-Coulter®, cat. no. A63881). We quantified cDNA using a Qubit® 2.0 fluorometer and Qubit® dsDNA High Sensitivity Assay kit (Invitrogen™, cat. no. Q32851) and verified that fragments were distributed in the 260-320 bp range using an Advanced Analytical® Fragment Analyzer™ (High Sensitivity Large Fragment Analysis Kit, Advanced Analytical®, cat. no. DNF‑493‑0500). Adapter dimers were removed with a 1.2X bead:sample volume clean‑up step. Two libraries were not constructed due to poor RNA quality and two libraries were lost during construction due to ethanol carryover for a total of 44 libraries.