Nebnext multiplex oligos for dual index primer set 1

Genomic DNA was sheared to a target fragment size of 350 bp length with the ultrasonicator Covaris LE220 following a standard protocol (30 μL volume, 220 W peak incident power, 89 s treatment time). Metagenomic libraries were prepared from 10 ng sheared DNA with the NebNext Ultra II DNA Library Prep Kit for Illumina. Sample-specific adaptations included tenfold adapter dilution, no size selection by adding 1 volume (89 μL) of Cytiva Sera-Mag Select beads in the first cleanup and eight PCR cycles in the amplification step. Nebnext Multiplex Oligos for Illumina (Dual Index Primer Set 1) were used for library multiplexing. The final cleanup was done with a left side size selection by adding 0.7 volumes (35 μL) of Cytiva Sera-Mag Select beads. A 1 nM library pool spiked with 3% PhiX was sequenced at the FGCZ using the NextSeq2000 platform and 2 × 150 bp PE-reads with a target fragment size of 500 bp, resulting in approximately 30,000,000 reads per sample.

RNA was isolated using RNeasy Kit (Qiagen). RNA was purified and washed via precipitation with 80% ethanol and quantified using NanoDrop 1000 (Peqlab). For reverse transcription and NGS library preparation, the NEBNext Ultra RNA Library Prep Kit for Illumina (E7530) and NEBNext Multiplex Oligos for Illumina, Dual Index Primer Set 1 (E7600) were used according to the manufacturer’s instructions. DNA fragments and index PCR products were purified with HighPrep (MagBio) beads. The sequencing library concentration was determined using the Quant-iT PicoGreen dsDNA Assay-Kit (Thermo).

Total RNA was extracted with TRIzol reagent (Sigma) according to the manufacturer's protocol and DNase I (NEB) was added to remove remaining DNA. 1 μg of purified RNA was processed according to the manufacture protocol of the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (E7530) and with the NEBNext® Multiplex Oligos for Illumina® (Dual Index Primer Set 1) (E7600). Libraries were sequenced on the Illumina Novaseq 6000 instrument. Mapping of fastq files was performed with STAR (50 (link)) and differentially expressed genes were identified using EdgeR (51 (link)). The RNA sequencing data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE213892 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213892).