Jagodinsky et al 2024 bulk RNA-seq counts

On day 3 following BT, tumors were collected and tissue samples were taken from the high, moderate, and low dose regions. The samples were homogenized in Trizol reagent (ThermoFisher) using a Bead Ruptor Elite (OMNI). RNA was isolated using Qiagen’s RNeasy Mini Kit according to the manufacturer’s instructions. 1 µg of RNA was used for library preparation (Illumina) with UD indexing (Illumina) according to manufacturer’s instructions. The prepared libraries were sequenced on an Illumina NOVAseq 6000. Skewer was used to trim adapter sequences from reads and to remove low quality bases from read 3’ ends before aligning to GRCm38.p6 using STAR.

RSEM was used to calculate expected gene counts from aligned reads. Principal component analysis and Pearson correlation between vectors of gene counts that belong to biological replicates was employed to detect outlier libraries. Any samples with inter-replicate Pearson correlation less than 0.9 or which did not cluster with replicates by PCA were dropped from downstream analyses.