title: 3'mRNA-Seq of drug-tolerant medulloblastoma cell lines.
design: 3'mRNA-Seq of drug-tolerant medulloblastoma cell lines.
library_construction_protocol: Drug-tolerant cell lines were pelleted and snap frozen in liquid nitrogen. Samples were stored at -80oC until downstream use. RNA was isolated utilising the NucleoSpin® RNA Plus kit (Machery-Nagel) as per the manufacturer's instructions, with an elution volume of 40 μL. RNA concentration and purity was analysed using a Nanodrop2000 spectrophotometer (Thermo Scientific) and only samples which met the following criteria were submitted for library preparation: RNA concentration > 100 ng/μL; 260/280 ratio 1.9 – 2.1; 260/230 ratio 1.8 – 2.2. Further quality control was undertaken using an Agilent TapeStation 4200 with the Agilent RNA ScreenTape Assay Kit (Agilent; 5067-5576 and 5067-5577) to test RNA integrity levels and a Qubit Fluorometer with the Qubit RNA BR Assay Kit (ThermoFisher Scientific; Q10211) to assess RNA quantity. 3'mRNA sequencing (3'mRNA-Seq) libraries were prepared using the QuantSeq 3' mRNA-Seq library prep kit for Illumina in the forward read direction supplemented with unique molecular index (UMI) as per manufacturer's instructions (Lexogen; 015.96). The aforementioned kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed. For each sample, cDNA was generated from 250 ng total RNA. First, total RNA was reverse transcribed using oligo-dT primers containing an Illumina-specific linker sequence. First-strand synthesis and RNA removal was followed by random-primed synthesis of the complementary strand. The random primer contained another Illumina-specific linker sequence. The resulting double-stranded cDNA was purified with magnetic beads. Library PCR amplification then introduced the complete adapter sequences required for cluster generation and UMIs which allow elimination of possible PCR duplicates in sequencing datasets (i7 6nt Index Set; 015.96; Lexogen). 14 PCR cycles were found to be optimal for library amplification. Libraries were next quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (ThermoFisher Scientific; Q32854), and library fragment-length distributions analysed using the Agilent TapeStation 4200 and the Agilent High Sensitivity D1000 ScreenTape Assay (Agilent; 5067-5584 and 5067-5585). Finally, libraries were pooled in equimolar amounts and final library quantification performed using the KAPA Library Quantification Kit for Illumina (Roche; KK4824).