Nextseq 500 mid output flow cell

A cDNA library of the CLIP samples was prepared using an NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300; NEB) in accordance with the manufacturers’ instructions with certain modifications. First, a 3′ SR adapter and 5′ SR adapter (Illumina) were diluted 10-fold with nuclease-free water before use. Second, each reagent was prepared at half the volume instructed by the manufacturer. Third, 2.5 μl of each purified RNA was used for cDNA library preparation. PCR amplification was performed for cDNA under the following conditions: 18, 20, and 22 cycles for initial amplification and 23 cycles during the final amplification. cDNA libraries were quantified using a Qubit double-stranded DNA (dsDNA) assay (Q32854; Invitrogen) and Agilent 2100 Bioanalyzer dsDNA assay (Agilent). High-throughput sequencing was performed at Core Unit Systems Medicine, University of Würzburg, Würzburg, Germany. Twelve cDNA libraries from CLIP were pooled on an Illumina NextSeq 500 mid-output flow cell and sequenced in paired-end mode (2 × 75 cycles). For total RNA sequencing, high-throughput sequencing was performed at Vertis Biotechnologie AG, Freising, Germany. cDNA libraries were prepared from total RNA and pooled on an Illumina NextSeq 500 mid-output flow cell and sequenced in single-end mode (1 × 75 cycles).

Total RNA was extracted from parental and resistant RPMI-8226 and L-363-cells in triplicate using the QIAGEN RNeasy mini kit and included the optional digestion with RNAse-free DNAse I (QIAGEN, Hilden, Germany). RNA quality was analyzed on Agilent Bioanalyzer RNA nano chips (Agilent Technologies, Inc., Santa Clara, CA, USA) and revealed RIN values between 9.5 and 9.9. An amount of 50 ng of total RNA was used to generate NGS libraries using the QuantSeq 3′ mRNA FWD kit (Lexogen, Vienna, Austria), which generates libraries of sequences close to the 3′ end of poly(A) RNAs. Libraries with unique barcodes were pooled, quantitated by qPCR and subjected to single-read sequencing on a NextSeq 500 MidOutput flowcell (Illumina, San Diego, CA, USA) as recommended by the manufacturer. After demultiplexing, reads were subjected to the automated QuantSeq data analysis pipeline provided by Lexogen. In a first step, the pipeline performed trimming, quality control, alignment, and read counting from fastqc input. Thereafter, differentially expressed genes were identified by the differential expression (DE) QunatSeq pipeline (Lexogen, Vienna, Austria).

cDNA libraries of RNA-seq-samples were constructed by Vertis Biotechnology AG, Munich, Germany. To deplete ribosomal transcripts, RNA-seq samples were treated with the Ribo-Zero “Bacteria” kit (Illumina) followed by RNA fragmentation and adapter ligation. UV CLIP-seq libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (#E7300, New England Biolabs) according to the manufacturer’s instructions as described before^{3 (link)}. cDNA libraries of RNA-seq samples were pooled on an Illumina NextSeq 500 high‐output flow cell and sequenced in single-read mode (1 × 76 cycles). cDNA libraries of UV CLIP samples were pooled on an Illumina NextSeq 500 mid‐output flow cell and sequenced in paired-end mode (2 × 75 cycles). The raw, de-multiplexed reads as well as the normalized coverage files of all cDNA libraries have been deposited in the National Center for Biotechnology Information’s Gene expression omnibus (GEO) and are accessible via the GEO accession number GSE129868. Statistics on cDNA library sequencings are summarized in Supplementary Tables 5 and 6.