Truseq small rna sample library preparation kit

RNASeq was used to analyse the miRNA and mRNA expression in each CSF pool. Small RNASeq was performed using 3.67 ng of RNA in the Illumina TruSeq small RNA sample library preparation kit (RS-200-0012), as previously reported [7 (link)], with reagents used in a half-reaction. Samples were assigned one of 48 possible indices, and went through 16 PCR cycles. Indexed samples were run on a gel and purified away from the adaptor band. The samples were then pooled and placed on a single-read Illumina V3 flowcell (GD-401-3001). Long RNASeq was performed using 2 ng of RNA in a NuGen Ovation RNASeq FFPE system (7150) for cDNA synthesis and RNA amplification. The samples were quantified using the Qubit dsDNA HS Assay Kit (Q3285; ThermoFisher), then moved forward to a KAPA Hyper Library Preparation Kit (KK8502; KAPA Biosystems). Each sample was assigned one of eight possible indices after one cycle of PCR and final libraries were quantified using a KAPA SYBR FAST Universal qPCR Kit (KK4824; KAPA Biosystems). Pooled and paired libraries were placed on a Paired End Illumina V3 flowcell (FPE-401-3001; Illumina). A threshold value of ≥5 counts per RNA was used as the cut-off value for inclusion in the small (miRNA) and long (mRNA) RNASeq data analysis.

miRNA sequencing was carried out by Genesky Bio-Tech (Shanghai, P.R. China). Total RNA was isolated and purified from RELA overexpressing or control A549 cells. At least 2 μg of qualified RNA samples with a high integrity number (RIN ≥ 7.0) was used for library construction using the TruSeq small RNA sample library preparation kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina HiSeq 2500 sequencing system (Illumina). The MiRDeep2 software was used to analyze miRNAs and their expression data. Differential gene expression analysis was estimated with the DESeq2 Bioconductor software package. p < 0.05 and log₂ fold change (FC) ≠ 0 were defined as the differential expression of miRNA between the two groups, in which log₂FC > 0 was labeled up and log₂FC < 0 was labeled down.