Project description:Purpose: Next-generation sequencing (NGS) is the main way to mine ncRNA. The purpose of this study is to screen out tRNA-derived small RNAs (tsRNAs) that are differentially expressed in proliferative and quiescent HASMCs Methods: tsRNAs profiles of proliferative and quiescent HASMCs were generated by deep sequencing, in triplicate, using Illumina NextSeq 500. The sequence reads that passed Illumina chastity filter were used for the following analysis. Trimmed reads (with 5ʹ, 3ʹ-adaptor bases removed) were aligned to mature-tRNA and pre-tRNA reference sequences. The alignment statistical analysis was applied to retain the valid sequences for subsequent tsRNAs expression profiles and differential expression analysis. Results: Using an optimized data analysis workflow, we mapped about 8 million sequence reads per sample to mature-tRNA and pre-tRNA sequences from GtRNAdb and identified 3,891 tsRNAs in the proliferative and quiescent HASMCs. According to the standardized TPM, 887 up-regulated and 951 down-regulated tsRNAs were found in the proliferative HASMCs (fold change > 2, P < 0.05).
Project description:Small RNA (<100 bases in length) were purified from total RNA using miRNeasy kit (Qiagen), then sequenced. Libraries were prepared at the Genomics Platform of the Cochin Institute, following the TruSeq small RNA protocol (Illumina), starting from 1 µg of high quality total RNA. Single read (1 × 75 bp) sequencing was performed on a Nextseq 500 platform (Illumina). FASTQ sequences were aligned on miRBase v.2052, then counted with STAR (v.2.5.2a). Counts were normalized with DESeq v1.30.0
Project description:A knockout cell library in Huh7.5.1 cells was generated by introducing a genome-scale CRISPR library (GeCKOv2, Addgene #1000000049) and subjected to hepatitis A virus infection (HM175/18f) to isolate virus-resistant mutant cells. Genomic DNA was isolated from the original and virus-selected mutant cell populations and abundance of guideRNA encoding sequences were measured by sequencing on an Illumina NextSeq (High Output).
Project description:Purpose: Determine if alveolar macrophage express different transcriptional programs when CD44 expression is lost. Methods: Alveolar macrophages were isolated from the bronchoalveolar lavage of 6-10 week old CD44+/+ and CD44-/- female mice. RNA was isolated an sequenced using Illumina NextSeq 500. RNA was sequenced by the UBC Biomedical Research Center. RNA quality, 18S and 28S ribosomal RNA with RIN = 9.6, was determined by Agilent 2100 Bioanalyzer following standard protocol for NEBnext Ultra ii Stranded mRNA (New England Biolabs). Sequencing was performed on the Illumina NextSeq 500 with paired end 42bp 42bp reads. De-multiplexed read sequences were then aligned to the Mus musculus (mm10) reference sequence using Spliced Transcripts Alignment to a Reference, STAR (https://www.ncbi.nlm.nih.gov/pubmed/23104886), aligners. Results and conclusion: Multiple pathways were abnormal in CD44-/- alveolar macrophages, in particular those involved in lipid metabolism and immune signaling.