Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Whole transcriptome RNAseq of the effect of photobiomodulation after spinal cord injury

ABSTRACT: The purpose of the experiment was to determine whole transcriptome changes after spinal cord injury (SCI) to understand how photobiomodulation promotes neuroprotection and functional recovery. SCI was performed at the thoracic (T) level T8, by crushing the dorsal columns using a calibrated watchmaker's forceps. Animals were then treated with photobiomodulation (PBM; 660nm wavelength) or sham control light, within 15 minutes of the injury. PBM or sham-treatment was treatment directed at the lesion site for 1 minute duration every 24hr for the first 3 days. Animals were then killed, tissues harvested and the RNA was extracted using RNEasy lipid tissue mini kit (Qiagen) according to the manufacture's instructions. Whole genome sequencing was outsourced to Qiagen RNA Sequencing Services (Germany).Library preparation was performed using the QIAseq Stranded Total RNA Library Kit with QIAseq FastSelect rRNA and globin depletion. QIAseq FastSelect rRNA HMR was used to reduce the amount of unwanted RNA species. After first and second strand synthesis, the cDNA was end-repaired and 3’ adenylated. Sequencing adapters were ligated to the overhangs. Adapted molecules were enriched using 16 cycles of PCR and purified by a bead-based cleanup. Library preparation was quality controlled using capillary electrophoresis (High Sensitivity Tape D1000) and high-quality libraries were pooled based on equimolar concentrations. The library pool(s) were quantified using qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing on a NovaSeq (Illumina Inc., Madison, USA) instrument (2x75, 2x10) according to the manufacturer instructions (Illumina Inc.). Raw data was de-multiplexed and FASTQ files for each sample were generated using the bcl2fastq software v2.20.0.422 (Illumina inc.).

INSTRUMENT(S): none, non, Illumina

ORGANISM(S): Rattus norvegicus

SUBMITTER: Zubair Ahmed

PROVIDER: E-MTAB-14593 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The objective of the study was to understand gene expression patterns and pathways that relate to Endoscopic Remission and Histologic remission in IBD as defined by different endoscopic and histological disease activity scores. Colonic Endoscopic biopsies were collected in RNAlater from 21 UC patients undergoingcolonoscopies withthe endocytoscope CF- Y-0058-1 prototype Olympus, Japan). RNA was extracted using RNeasy columns (Qiagen) and quantified by Qubit (Life Technologies). 0.5ng RNA was used to prepare uniquely indexed cDNA QIAseq UPX 3’ Transcriptome libraries according to manufacturer’s instructions (Qiagen). Libraries were quantified and quality-controlled using the QIAseq Library Quant Assay Kit and tapestation analysis and sequenced on the Miseq and Nextseq Illumina platforms using the illumina sequencing primer and PhiX 15% to a depth of 1-3milion reads/sample. Read numbers were set as 101 bp (R1) x 51 bp (R2). Fastq files were obtained through BaseSpace and reads de-multiplexed, aligned to human genome GRCh38, quantified and normalised by the TPM method using the CLC Genomics Workbench (Qiagen). The following differential expression procedure was performed for participants classified as healed versus non-healed according different histologic and endoscpic scores which included the histological scores: Nancy and Robarts Histological Index (RHI) as well as the mayo endoscopic score (MES) and the endocytoscopy score system (ECSS). Normalised values were log-transformed with a pseudocount of 1 added. Transcripts with < 1 transformed count in any sample were excluded from further analysis, as were transcripts with low variance (defined as less than 10% unique counts across both conditions and greater than a 19:1 ratio of the most frequent count to the second most frequent across both conditions). Differential expression analysis between conditions was conducted with the limma package. Library size was estimated using reduced maximum likelihood estimator with 500 iterations. Initial fitting was performed using a robust M-estimation, and moderated test statistics computed by empirical Bayes. A FDR corrected p value <0.05 was considered and filtered for further downstream data analysis. Partial least squares discriminant analysis (PLS-DA) modelling was performed on these genes and a variable Importance in Projection (VIP) score of >1 was used to further filter the genes. To understand the biological significance and pathways, we also performed enrichment analysis using EnrichR involving three databases: Gene ontology, Wiki pathways and Kyoto Encyclopaedia of Genes and Genomes (KEGG).

Project description:The objective of the study was to understand gene expression patterns and pathways that relate to Endoscopic Remission and Histologic remission in IBD as defined by different endoscopic and histological disease activity scores. Colonic Endoscopic biopsies were collected in RNAlater from 9 Crohn's disease patients undergoing colonoscopies with the endocytoscope CF- Y-0058-1 prototype Olympus, Japan). RNA was extracted using RNeasy columns (Qiagen) and quantified by Qubit (Life Technologies). 0.5ng RNA was used to prepare uniquely indexed cDNA QIAseq UPX 3’ Transcriptome libraries according to manufacturer’s instructions (Qiagen). Libraries were quantified and quality-controlled using the QIAseq Library Quant Assay Kit and tapestation analysis and sequenced on the Miseq and Nextseq Illumina platforms using the illumina sequencing primer and PhiX 15% to a depth of 1-3milion reads/sample. Read numbers were set as 101 bp (R1) x 51 bp (R2). Fastq files were obtained through BaseSpace and reads de-multiplexed, aligned to human genome GRCh38, quantified and normalised by the TPM method using the CLC Genomics Workbench (Qiagen). The following differential expression procedure was performed for participants classified as healed versus non-healed according different histologic and endoscpic scores which included the histological scores: Nancy and Robarts Histological Index (RHI) as well as SES-CD, Modified Riley and and the endocytoscopy score system (ECSS). Normalised values were log-transformed with a pseudocount of 1 added. Transcripts with < 1 transformed count in any sample were excluded from further analysis, as were transcripts with low variance (defined as less than 10% unique counts across both conditions and greater than a 19:1 ratio of the most frequent count to the second most frequent across both conditions). Differential expression analysis between conditions was conducted with the limma package. Library size was estimated using reduced maximum likelihood estimator with 500 iterations. Initial fitting was performed using a robust M-estimation, and moderated test statistics computed by empirical Bayes. A FDR corrected p value <0.05 was considered and filtered for further downstream data analysis. Partial least squares discriminant analysis (PLS-DA) modelling was performed on these genes and a variable Importance in Projection (VIP) score of >1 was used to further filter the genes. To understand the biological significance and pathways, we also performed enrichment analysis using EnrichR involving three databases: Gene ontology, Wiki pathways and Kyoto Encyclopaedia of Genes and Genomes (KEGG).

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Dataset Information

Whole transcriptome RNAseq of the effect of photobiomodulation after spinal cord injury

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