Sample07
Library preparation and sequencing were conducted at the Texas A&M Institute for Genome Science and Society. Ribosomal RNA depletion was not conducted for this experiment due to low RNA extraction yields. Extracted RNA was quantified and evaluated for degradation with an Agilent Technologies 2200 TapeStation using the High Sensitivity RNA ScreenTape System protocol with 2 L of RNA extract (Agilent Technologies Inc., CA, USA). Library preparation for RNAseq was conducted according to the TruSeq Stranded Total RNA protocol (Illumina Inc., CA, USA) with slight modification. Library preparation began with adding 8.5 L of extracted RNA to the DFP (Depleted RNA Fragmentation Plate) plate with no prior cleaning steps. No control was used for the library preparation and resuspension buffer (RSB) was substituted where indicated. The final library was quantified with a Qubit 2.0 Fluorometer (ThermoFisher Scientific Inc., MA, USA) and with an Agilent 2200 TapeStation for bp size information. The nM concentration was calculated and samples were normalized in a two-step process. Samples were first normalized to 10 nM, followed by normalization to 4 nM in low EDTA. Final 4 nM samples were pooled and sent to the North Texas Genome Center for sequencing with an Illumina NovaSeq 6000.