Nebnext ultra 2 directional rna library prep

Bacterial and human rRNA were depleted from total RNA using NEBNext rRNA Depletion Kits E7850 and E7400 (New England Biolabs), respectively. A cocktail of human and bacterial depletion solutions was used in the ratio of 2:1. Depleted RNA was purified using Agencourt RNA clean XP beads (Beckman Coulter) and used for preparation of RNA sequencing (RNA-seq) libraries using NEBNext Ultra II Directional RNA Library Prep (New England Biolabs) as per the manufacturer's protocol. The RNA fragmentation time was optimized and adjusted according to RIN no. Libraries were labeled with unique indexes for multiplexing using NEBNext Multiplex Oligos for Illumina (New England Biolabs). The final libraries were quantified using Qubit dsDNA HS Assay Kit on Qubit 3.0 fluorimeter. The library quality and size distribution were assessed using Agilent High sensitivity DNA kit on Bioanalyzer 2100. Library concentrations are presented in Supplementary Data S1. The 60 libraries were pooled in groups of 10 and sequenced using 2 × 100 CoolMPS chemistry on DNBSEQ-T7 platform (BGI) with a target depth of 400 million paired reads per sample (brute-force deep sequencing).

For library preparation, mRNA was isolated from DNAse-treated total RNA using the NEBNext rRNA depletion Kit (human, mouse, rat) from New England Biolabs (NEB) according to the manufacturer’s instructions. Final elution was done in 5 μl nuclease-free water. The samples were then directly subjected to the workflow for strand-specific RNA-Seq library preparation (NEBNext Ultra II Directional RNA Library Prep; New England Biolabs). For ligation, custom adaptors were used (Adaptor-Oligo 1: 5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3′, Adaptor-Oligo 2: 5′-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3′). After ligation, the adapters were depleted by XP bead purification (Beckman Coulter) adding beads in a ratio of 1:0.9. Dual indexing was done during the following PCR enrichment (15 cycles, 65°C) using custom amplification primers carrying the index sequence indicated with “NNNNNNN.” (Primer1: AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, primer2: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T). After two more XP bead purifications (1:0.9), libraries were quantified using the Fragment Analyzer (Agilent). Libraries were equimolarly pooled before sequencing them with a length of 75 bp in single end mode on an Illumina NextSeq 500 system to a depth of at least 40 mio reads.