Similar protocols

Pipeline publication

[…] using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs Inc., Frankfurt, Germany). Adaptors and primers were diluted 1:2 in nuclease-free water to accommodate the low RNA input. Size selection of pooled PCR products was performed by agarose gel electrophoresis (4%), cutting out bands with 130 to 150 bp fragments. The purity and concentration of the libraries extracted from the gel were verified by capillary electrophoresis using the High Sensitivity DNA kit on the Bioanalyzer 2100 (Agilent Technologies). Finally, the libraries were subjected to Illumina single-end sequencing-by-synthesis using 50 cycles on the HiSeq 2500 (Illumina Inc., San Diego, CA, USA)., FastQC (version 0.11.5) was used to assess the sequence length distribution and quality of the NGS data, as previously described (). Adaptor sequences were trimmed using BTRIM, and all reads without adaptors were discarded (). Additionally, reads shorter than 15 nt were excluded from the data set (). Prior to miRNA analysis, reads pertaining to ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA) were removed by mapping to sequences obtained from RNAcentral (). The remaining reads were then aligned to miRBase (version 21) (). Mapping was carried out using Bowtie and the 'best' alignment algorithm, allowing one mismatch for both RNAcentral and miRBase (). Final read count tables were generated by sorting and indexing aligned reads using SAMtools, and calling the sum of hits per miRNA sequence (). Differential gene expression analysis was subsequently performed via the bioconductor package DESeq2 (version 1.8.1), using the Benjamini-Hochberg method to correct for false discovery (). A log2 fold change ≥|1| and an adjusted p-value (Padj) of ≤0.05 were set as thresholds to identify significantly regulated miRNAs. Only miRNAs with a mean expression of at least 50 counts were included in the analysis. Principal component analysis (regularized log-transformed, sizefactor-corrected counts obtained from DESeq2), and data visualization were performed in R (version 3.4.0) using the packages gplots, ggfortify, genefilter and RColorBrewer., Based on the NGS data, differentially regulated cellular miRNAs were validated by RT-qPCR. First, 111 ng of RNA were reverse transcribed in triplicate using the miScript II RT kit (Qiagen) according to the manufacturer's instructions. A total of 1 µl cDNA was subjected to real-time PCR in a 10 µl reaction volume using the miScript SYBR-Green PCR kit (Qiagen). Reactions were run on a CFX384 real-time PCR detection system (Bio-Rad) using the recommended protocol of polymerase activation (95°C for 15 min) and 45 cycles of amplification (94°C for 15 sec, 55°C for 30 sec, 70°C for 30 sec). Quantification cycle (Cq) values were determined automatica […]

Pipeline specifications

Software tools FastQC, Btrim, Bowtie, SAMtools, DESeq2, gplots
Databases RNAcentral
Organisms Homo sapiens
Diseases Breast Neoplasms, Breast Diseases, Neoplasm Metastasis, Neoplastic Processes, Neoplasms