GSM9509448: RNAseq, PHH Donor3, PCSK9-EE7000, 3; Homo sapiens; RNA-Seq
For RNA extraction of all donors and sample types, Beckman Coulter RNAdvance v2 Kit was used. For RNA extraction from each PHH donor, two 24-well replicate plates per donor containing 336 µL cell lysate were combined and 4 x 63 µL aliquots of lysate were processed . For RNA extraction from each HAdCC donor, all replicate wells of each condition from two replicate plates each containing 3 wells of 63 µL cell lysate were combined and 3 x 63 µL aliquots of lysate were processed. For RNA extraction from HSEC, all replicate wells of each condition from two replicate plates each containing 3 wells of 63 µL cell lysate were combined and 3 x 63 µL aliquots of lysate were processed. The remaining lysate from each cell type was used for DNA extraction. For all samples, lysate aliquots were transferred to separate technical replicate wells of a 0.5 mL deep well plate and mixed with Beckman Coulter-patented SPRI paramagnetic beads to bind total RNA from the cell lysate. Bound RNA was then pelleted with a plate magnet, removing the supernatant allowing DNA and other contaminants to be removed through DNase treatment and a series of buffer (Wash WBD) and 70% ethanol washes, respectively. Total RNA was then eluted from the magnetic beads in nuclease-free water (HAdCC and HSEC samples (96-well plate format) were eluted in 22 uL per sample combining the replicates for a total of 44 uL eluate. PHH samples (24-well format) were eluted in 26 uL.). Total RNA sequencing libraries were generated using the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus. Samples were normalized with nuclease-free water to 100 ng in 11 µL total volume. Concentrations of final libraries were determined by using either the Quant-iT High Sensitivity DNA kit on the VICTOR Nivo plate reader or the Qubit DNA High Sensitivity kit on the Qubit Flex Fluorometer. Library fragment size was also checked on a DNA D1000 or HSD1000 ScreenTape on the Agilent 4200 Tapestation instrument. To ensure proper quantification of sequenceable library molecules, a qPCR reaction was also performed using the NEBNext Library Quant Kit for Illumina.  Total RNA-seq libraries were shipped to SeqCenter for sequencing on a NovaSeq X. Sequencing used a 2 x 151 bp configuration and targeted sequencing depth was >100 M read-pairs per library. Note that PHH Donor 3 sequencing data had a high percentage of duplicates. Therefore, the library prep and sequencing were repeated, with RNA-seq libraries sequenced in the Sponsor's laboratory on a NextSeq2000 with a 2 x 151 bp configuration and targeted sequencing depth >100 M read-pairs per library.