Ribocop kit

Suboptimal performance of commercial depletion kits in Ribo-seq is a known issue (Chung et al., 2015 (link)) and we provide evidence on this by analyzing two public datasets (Simsek et al., 2017 (link); Zinshteyn et al., 2020 (link)), in addition to one experiment performed here using the RiboCop kit (Lexogen, catalog no. 037). We accessed the datasets through NCBI using the GSE147324 (Zinshteyn et al., 2020 (link)) and GSE96998 (Simsek et al., 2017 (link)) ids. For the former, adapter trimming was performed with given instructions (Zinshteyn et al., 2020 (link)), and, for the latter, cutadapt tool (Martin, 2011 ) was used with the following parameters, –action=trim –discard-untrimmed -m 18 –adapter CTGTAGGCACCATCAAT -O 15 –error-rate = 0.1. The rRNA fragments were then identified through mapping the trimmed reads to human and mouse 28S, 18S, 5.8S and 5S rRNA sequences, using TopHat (Kim et al., 2013 (link)) with the following parameters, –no-novel-juncs –no-novel-indels –no-coverage-search. We also mapped them to protein-coding transcripts (gencode v34 and vM21) after cleaning rRNA fragments using the SortMeRNA tool (Kopylova et al., 2012 (link)), and calculated the rRNA percentages by dividing the number of rRNA-mapping reads by the total number of reads that maps to rRNAs or protein coding transcripts.

PDX samples were taken from storage at −80°C and broken apart in liquid nitrogen using a mortar and pestle. The tumor fragments were then weighed in as to use at least 20 mg of tumor tissue and homogenized in either TRIzol reagent using a rotor stator instrument or crushing in the mortar and pestle with buffer from the AllPrep DNA/RNA Mini Kit (QIAGEN, 80204). RNA was extracted either using the TRIzol (Invitrogen, 15596018) or AllPrep kits. Afterward, 500 ng of RNA per sample was processed using the RiboCop kit (Lexogen, 037) for rRNA removal.

RNA was isolated from sorted SSCs by using QIAzol Lysis Reagent (79306, Qiagen) and following the manufacturer’s instructions. To generate RNA-seq libraries total RNA was treated with DNAse I, in 10X Buffer (AMPD1, Sigma). RNA was purified using Rneasy MinElute columns (74204, Qiagen) and ribosomal RNA depletion was performed with the RiboCop kit (Lexogen). Ribo-depleted RNA was used to generate libraries with the SENSE Total RNA-Seq Library Prep Kit (Lexogen) following the manufacturer’s instructions. Libraries were sequenced with an Illumina HiSeq2500 on a 50 bp, single-end run.