title: RNA-seq of wild-type S rosetta + rpl36A[P56Q], rep 5
design: Library preparation and sequencing was performed by the QB3-Berkeley Genomics core labs (QB3 Genomics, UC Berkeley, Berkeley, CA, RRID:SCR_022170). 500 ng were of RNA were used for library prep using the KAPA mRNA capture kit (Cat. No. 07962240001) for poly-A selection and the KAPA RNA HyperPrep kit (Cat. No. 08105952001). Truncated universal stub adapters were ligated to cDNA fragments, which were then extended via PCR using unique dual indexing primers into full length Illumina adapters. RNA integrity was assessed by Agilent Bioanalyzer 2100 before library prep using an Agilent RNA 6000 Nano Kit (Cat. No. 5067-1511). Sequencing libraries were also confirmed by Bioanalyzer 2100 for the correct size distribution using the Agilent High Sensitivity DNA Kit (Cat. No. 5067-4626). Library concentration was quantified by qPCR using the KAPA Library Quantification Kit (Cat. No. 079601400001) and libraries were pooled at equal concentrations before sequencing. Sequencing was performed in one lane of an SP flow cell on the Illumina NovaSeq 6000 with an S4 flowcell, collecting150 bp paired-end reads. Reads were de-multiplexed using Illumina bcl2fastq2 (v 2.20) and default settings, on a server running CentOS Linux 7.