Pandey Lab, Johns Hopkins University School of Medicine
RNA-Seq of these six organs/tissues was performed according to the manufacturer's protocol using the Illumina TruSeq RNA Sample Preparation Kit and SBS Kit v3 (Illumina, San Diego, CA). Briefly, RNA quality was determined using an Agilent Bioanalyzer with an RNA Nano 6000 chip. RNA-Seq library construction was started using 500 ng of total RNA that was then subjected to poly(A)+ selection and fragmentation. Followed by first and second strand synthesis, the cDNA was subjected to end repair, adenylation of 3′ ends, and adapter ligation. One of six unique indices was used in each individual sample. After AMPure XP magnetic bead (Beckman Coulter, Brea, CA) clean-up, each cDNA sample was subjected to 15 cycles of PCR amplification using an ABI 9700 thermal cycler. The cDNA library quality and size distribution were checked using an Agilent Bioanalyzer with a DNA 1000 chip. Our libraries showed a size between 200 and 500 bp with a peak at ∼260 bp. All libraries were carefully quantitated using a Qubit 2.0 fluorometer (Invitrogen, Grand Island, NY) and were stored in microfuge tubes (Invitrogen) in a −20 °C freezer. The cluster generation was done using an Illumina TruSeq V3 flow cell with six different cDNA libraries with different indices in each lane, repeated in three lanes, at a concentration of ∼8.6 pm. RNA-Seq was carried out on Illumina's HiScanSQ system (Illumina) using the Illumina TruSeq SBS V3 sequencing kit and 50 bp by 50 bp paired reads.