GSM5531621: Subject 101694 LTA; Homo sapiens; RNA-Seq
RNA using the Qiagen RNeasy Mini kit (Germantown, MD) was used following the manufacturer's instructions. Briefly cells were lysed and homogenized in lysis reagent using QIAshredder homogenizers. 70% ethanol was added to the lysate prior to passing through a spin column at 8000 x g to remove genomic DNA. Spin columns containing RNA were washed with ethanol + Buffer RPE multiple times and centrifuged at 8000 x g, then RNA was eluted from the column in 30uL RNAse-free water. RNA was treated with DNase and concentrated (Zymo Research, Cat No R1014). RNA quality was assessed by bioanalyzer and all samples had RNA Integrity Numbers >6. QuantSeq 3'mRNA sequencing FWD (RNAseq) libraries (Lexogen, Cat 015.96) were prepared as stated in the Lexogen manual using 30ng of total input RNA per sample and 18 cycles of PCR amplification. 48 unique, dual index barcodes were used and samples were then pooled, exonuclease VII treated. Samples sequenced across three lanes of a HiSeq4000 to generate Single End 100 base pair reads. Fastq files were generated by concatenating reads across all three lanes for each sample.