Kapa library quantification kit

RNA was extracted from flash-frozen tumour tissue by homogenisation using sterile 1.5 mL tissue homogenizers and subsequent isolation using Qiagen RNeasy Mini Kit, according to manufacturer’s protocol (Qiagen, Germantown, MD, USA). Corresponding to the manufacturer’s requirements, 20–30 mg of tissue was used from each sample. Libraries of 3’mRNA were obtained from total RNA using the Lexogen QuantSeq kit according to standard protocol. After validation and quantification (2200 TapeStation, Agilent Technologies, Santa Clara, CA, USA and Qubit System, Invitrogen, Carlsbad, California, CA, USA respectively), pools of cDNA libraries were generated. Pools were quantified using the KAPA Library Quantification kit (Peqlab, Radnor, PA, USA) and the 7900HT Sequence Detection System (Applied Biosystems, Foster City, PA, USA) and lastly sequenced on an Illumina HiSeq4000 or NovaSeq6000 sequencer using a 1 × 50 base pair protocol.

Whole-exome sequencing was performed on 16 fresh frozen atypical carcinoids in the Cologne Centre for Genomics. Exomes were prepared by fragmenting 1 μg of DNA using sonication technology (Bioruptor, Diagenode, Liège, Belgium) followed by end repair and adapter ligation including incorporation of Illumina TruSeq index barcodes on a Biomek FX laboratory automation workstation from Beckman Coulter (Beckman Coulter, Brea, CA, USA). After size selection and quantification, pools of five libraries each were subjected to enrichment using the SeqCap EZ v2 Library kit from NimbleGen (44Mb). After validation (2200 TapeStation; Agilent Technologies, CA, USA), the pools were quantified using the KAPA Library Quantification kit (Peqlab, Erlangen, Germany) and the 7900HT Sequence Detection System (Applied Biosystems, Waltham, MA, USA), and subsequently sequenced on an Illumina HiSeq 2000 sequencing instrument using a paired-end 2 × 100 bp protocol and an allocation of one pool with 5 exomes/lane. The expected average coverage was approximately 120x after removal of duplicates (11 GB).

For DNA isolation, single strains were grown in a CGXII monoculture supplemented with 3 mM of the respective amino acid in the BioLector. From one well per mutant, gDNA was isolated with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Resulting gDNA concentration was determined via Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, USA). From the prepared gDNA, 1 µg was used for library preparation employing the NEBNext® Ultra™ II DNA Library Prep Kit (NEB, Frankfurt am Main, Germany). Via qPCR with the KAPA library quantification kit (Peqlab, Erlangen, Germany), the library was evaluated and then normalized via pooling. After in-house sequencing (paired-end sequencing via a MiSeq (Illumina®), read length of 2 × 150 bases), the demultiplexed fastq output files were processed with the CLC Genomic Workbench software (Qiagen, Hilden, Germany). For reads mapping and variants calling, the C. glutamicum ATCC 13032 reference genome BX927147 or the genome sequence of C. glutamicum C1 (CP017995) were used. Mutations and deletions were assessed manually regarding their specific occurrence between the different samples and their relevance. The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB60176 (https://www.ebi.ac.uk/ena/browser/view/PRJEB60176).

DNA extraction was performed from about 1 × 10⁶ cells using the Gentra Puregene Tissue Kit (Qiagen, Venlo, Netherlands) including RNAse treatment. DNA integrity was controlled by agarose gel electrophoresis prior to exome preparation from 200 ng of pooled DNA from 10 suppressor clones. Exome-enriched libraries for Illumina paired-end multiplexed sequencing were generated using the Agilent SureSelect^XT mouse all exon kit following the manufacturer’s recommendation on the automated Agilent Bravo liquid handling platform. After validation (2200 TapeStation, Agilent Technologies, Santa Clara, California) and quantification (Qubit System, Thermo Fisher Scientific) pools of libraries were generated and quantified using the KAPA Library Quantification Kit (Peqlab, Erlangen, Germany) and the 7900HT Sequence Detection System (Applied Biosystems, Foster City, California). Subsequent sequencing was performed on an Illumina HiSeq4000 sequencing instrument using a paired-end 2 × 75 bp protocol.

The TruSeq v2 RNA sample preparation kit (Ilumina) was used to prepare cDNA libraries from 200 ng total GFP+ or GFP− RNA. Poly (A)⁺ RNA was purified onto oligo-dT magnetic beads and was fragmented using divalent cations at elevated temperature; RNA fragments were reverse-transcribed using random primers, followed by second-strand cDNA synthesis with RNase H/DNA Polymerase I. After end repair and A-tailing, adapters were ligated and the indexed cDNA products were purified and amplified by PCR (15 cycles) to create the final cDNA libraries. Library quality was validated on a 2200 TapeStation (Agilent Technologies) and individual libraries were quantified on the Qubit System (Invitrogen) prior to pooling and pool quantification via the KAPA Library Quantification kit (Peqlab) and the 7900HT Sequence Detection System (Applied Biosystems). The pooled, indexed libraries were loaded and analysed on an Illumina GAIIx sequencer using the 2 × 100-bp v3 protocol.