Novaseq 6000 sequencing

Five strains of KPC-2-producing CRKP and five strains of CSKP were used for RNA-seq (Gene Denovo Biotechnology). Illumina NovaSeq 6000 sequencing and FASTP (https://github.com/opengene/fastp) software were used to obtain valid data.
The resulting data were compared with the reference genome assembly GCF_022869665.1 (https://www.ncbi.nlm.nih.gov/assembly/12472981) to obtain known genes by using Bowtie2 (version 2.2.8).
According to previous studies [31 (link),32 (link)], we annotated the unmatched genes with the NR database using Rockhopper. Unannotated genes with 50–500 nt of length and stable secondary structures were listed as candidate sRNAs [46 (link)]. In addition, we used the sRNAMap database (version 2009) and the Rfam database (version 13) to match known sRNAs.

In a pan-cancer cohort of 128 patients diagnosed according to ICD-O-3, RNA and DNA were isolated from fresh frozen tumor tissue and as a matching normal, DNA was isolated from whole blood. Blood and bone marrow samples were enriched for monocytic cells using Ficoll. Total RNA was isolated from tumor samples using the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN) according to standard protocol on the QiaCube (Qiagen). RNA-sequencing (RNA-seq) libraries were generated from 300 ng RNA using the KAPA RNA HyperPrep Kit with RiboErase (Roche) and sequenced with NovaSeq 6000 (2 × 150 bp) (Illumina). DNA was isolated from paired tumor-normal samples also using the AllPrep DNA/RNA/Protein Mini Kit. Whole-genome sequencing (WGS) libraries were generated from 150 ng DNA using the KAPA DNA HyperPlus kit and NovaSeq 6000 sequencing platform (Illumina).

Total RNA was collected in the frozen tissues of rhesus macaques with TRIzol reagent (Catalog No. 15596018, ThermoFisher Scientific, Waltham, MA). VAHTS Universal V6 RNA-seq Library Prep Kit (Catalog No. NR604, Vazyme, Nanjing, China) was used to construct libraries with the obtained RiboMinus RNA following the manufacturer’s instructions. Paired-end sequencing was performed on the Illumina NovaSeq 6000 sequencing system with 150 bp read length.

Total RNA was extracted from 22Rv‐1‐30e stable lines with and without doxycycline induction and used for RNA Sequencing performed by LC Sciences. First, RNA integrity was checked using Agilent Technologies 2100 Bioanalyzer (Agilant Technologies, Santa Clara, CA, USA). Ribosomal RNA was depleted, and the sequencing library was prepared using Illumina's TruSeq‐stranded‐total‐RNA‐sample preparation protocol (Illumina, San Diego, CA, USA). This includes RNA fragmentation, reverse transcription using random primers, dUTP incorporation, A‐ tailing, adapter ligation, strand degradation and PCR enrichment. Illumina's NovaSeq 6000 sequencing (Illumina) was used to perform paired‐end sequencing. RNA quality control and library preparation were performed by LC Sciences (Houston, TX, USA).

WBM were isolated from mouse tibiae, femora, and ilia leg bones by crushing in 2% FBS (PBS) buffer, add 1:1,000 DNase1 and then translate into 15 mL BD tube, centrifuge at 1,200 rpm, RT for 3 min. Then, the supernatant was discarded, 3 mL of 1x ACK lysis buffer was added to resuspend the bone marrow pellet on ice for 3 to 5 min, and PBS (more than lysis buffer) was added to terminate the lysis reaction. The supernatant was discarded, and 0.04% PBS-BSA buffer was added to resuspend the cell pellet, which was then filtered through a 40-μm cell strainer. More than 80% of the living cells in the sample and a density of living cells ranging from 600 to 1,200 cells/μl were preferable. Sample quality was measured using a Bio-Rad TC20 automated cell counter. Libraries were prepared using Chromium Single Cell 3′ Reagent Kits v2 according to the manufacturer’s protocol (10× Genomics) and were sequenced using Illumina NovaSeq 6000 sequencing.

A total amount of 5 μg RNA per sample was used as the input material for RNA sample preparations. First, ribosomal RNA was removed with an Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, Madison, WI, USA), and the rRNA-depleted RNA sample was cleaned with ethanol precipitation. Subsequently, sequencing libraries were generated using the cleaned RNA sample with a NEBNext^® Ultra™ Directional RNA Library Prep Kit for Illumina^® (NEB, Ipswich, MA, USA), according to the manufacturer’s instructions. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumia, San Diego, CA, USA). Afterwards, the libraries were sequenced by Illumina NovaSeq 6000 sequencing (150 bp*2, Shanghai BIOZERON Co., Ltd., Shanghai, China). The raw paired end reads were trimmed and quality controlled using Trimmomatic (version 0.36 http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic, accessed on 10 August 2021). Then clean reads were separately aligned to the reference genome with orientation mode using hisat2 (https://ccb.jhu.edu/software/hisat2/index.shtml, accessed on 10 August 2021) software.