title: Ribo-Seq of human A549 cells
design: Ribosome profiling lysates were clarified, RNase I treated and fractionated by sucrose cushion. RNA was extracted by Trizol extraction and size selected to obtain 25-34 nt protected fragments. Protocol used published in McGlincy and Ingolia, 2017. Extracted RNA was ligated to a universal DNA linker and rRNA depleted. Next, RNA was reverse transcribed, circularized and PCR amplified to generate libraries. Protocol used published in McGlincy and Ingolia, 2017. Reads were demultiplexed and trimmed up to UMI (unique molecular identifier) sequences. Reads were mapped against human non-coding RNA genes (including rRNA and tRNA) with bowtie1 and non-matching reads were retained, demultiplexed and UMIs were trimmed. Next, reads were aligned against the MANE v1.4 transcriptome using bowtie1 to obtain SAM files. SAM files were randomly downsampled since small files needed for purpose of software testing. The code run was: samtools view -h 80S_full.SAM | awk 'BEGIN {srand()} /^@/ {print} !/^@/ {if (rand() <= 0.5) print}' > 80S.SAM. The FASTQ files were obtained from SAM files using samtools, respresenting the subsetted cohort.