Nebnext rrna depletion kit human mouse rat

20–100 ng of FFPE RNA and paired fresh frozen RNA was used for library preparation using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs Inc., Ipswich, MA), following the manufacturers protocol for highly degraded (RIN ≤ 2) or intact (RIN > 7) samples respectively. Fragmentation is based on RIN value of RNA input and conducted as outlined in the protocol. Fragmentation for FFPE RNA was not performed. Experimental FFPE RNA and paired fresh frozen RNA from the same patient was used if available using similar input amounts for each sample type. A total of 13 libraries were prepared, including six patient pairs. Libraries were quality assessed using a combination of the Qubit dsDNA HS Assay (Invitrogen), the Bioanalyzer DNA 7500 Assay (Agilent Technologies), and KAPA Library Quantification Kit for Illumina (KAPA Biosystems, Boston, MA). Libraries were combined in equal molar amounts and sequenced across three lanes of an Illumina NextSeq 500 High Output flowcell using 75 × 2 bp paired end reads. Each flowcell generated a minimum of 800 million reads passing filter.

First, 200 ng of purified RNA were depleted for ribosomal RNA via NEBNext rRNA Depletion Kit (Human/Mouse/Rat) before library preparation via NEBNext Ultra II Directional RNA Library Prep Kit for Illumina as per manufacturer protocols. Final libraries were sequenced on NextSeq2000 in paired-end 100 bp configuration to a mean of 100-million paired reads per sample. Samples were demultiplexed and converted to fastq using bcl2fastq2 (Version 2.20). For the SingScore analysis, RNA-sequencing reads were first aligned to the human transcriptome (GRCh38; transcript annotation from Ensembl v109), and expression values were estimated as transcripts per million (TPM) using Salmon 1.9.0 [26 (link)], as implemented in the nf–core–rnaseq pipeline v-3.10.1 [27 (link)]. These values were aggregated to per-gene TPM, and any genes that did not align more than 10 read pairs in at least one sample were excluded from further analysis. SingScore rank scores for the 18-gene interferon–gamma gene signature [28 (link)] were then calculated from the per-gene TPM values using SingScore v.1.16.0 [29 (link)]. The list of genes analysed included: CD3D, IDO1, CIITA, CD3E, CCL5, GZMK, CD2, HLA-DRA, CXCL13, IL2RG, NKG7, HLA-E, CXCR6, LAG3, TAGAP, CXCL10, STAT1, and GZMB.

Total RNA was extracted from collected spermatid fractions with Monarch RNA Extraction Kit (NEB) according to the manufacturer’s protocol for RNA extraction from tissue or leukocytes, with proteinase K incubation time optimized for homogenized tissues. The optional step with DNase I treatment was performed, with an additional treatment with Turbo DNase to eliminate all traces of genomic DNA (Invitrogen). Total RNA was then purified with Monarch RNA Cleanup Kit (NEB) and quantified using Qubit Fluorometric Quantification (Thermo Fisher Scientific). The integrity of the RNA was determined using the TapeStation High Sensitivity RNA kit (Agilent Technologies). 10 ng of total RNA has been used to prepare ribo-depleted RNA-seq libraries with these kits: NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. We used 17 PCR cycles to obtain enough material for sequencing. Obtained NGS libraries were pooled in equimolar amounts and sequenced using the Illumina NextSeq 500 sequencer with a 40PE running mode.

Approximately 1 μg of total RNA eluted in RNase-free water was sent to SEQme s.r.o (Dobris, Czech Republic) for RNA library construction and deep sequencing. The rRNA was depleted with an NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and constructed with an NEBNext Ultra II Directional RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (Unique Dual Index Primer Pairs) (NEB, Ipswich, MA, USA). Library QC was assessed in an Agilent Bioanalyzer 2100 High sensitivity DNA Kit. A KAPA Library Quantification Kit for Illumina platform was used for absolute, qPCR-based quantification of the Illumina libraries flanked by the P5 and P7 flow cell oligo sequences. Libraries underwent paired-end (PE) (2 × 150 nt) sequencing on a NovaSeq6000 (DS-150) (Illumina, San Diego, CA, USA) using a NovaSeq S4 v1.5 reagent kit. An “in-lane” PhiX control spike was included in each lane of the flow cell.