Genome_build: Saccharomyces cerevisiae genome (R64-1-1) In CLC Genomics Workbench, the ‘Differential Expression for RNA-Seq tool’ performs some multi-factorial statistics on a set of Expression Tracks based on a negative binomial Generalized Linear Model (GLM). In this study, genes that were considered differentially regulated met the following criteria: FDR p-value ≤0.05 and fold change ≥ 2 for up regulated or ≤ 0.5 for down regulated genesįor RNA Seq, each study group contained three library samples. The threshold p-value was determined according to the false discovery rate (FDR). RNA-Seq data were analyzed by using the CLC Genomics Workbench (Qiagen Bioinformatics) to identify the differentially expressed genesĮxpression values were measured in RPKM (Reads per kilobase of exon model per million mapped reads). DNA fragments with 300-450 bp inserts were selected for massive sequencing. Specific adapters for Illumina sequencing were attached to the cDNAs and the library was enriched by limited PCR. Briefly, depleted RNA was fragmented and copied to double-stranded cDNA. RNA purity and integrity were assessed using RNA Nano Labchips in an Agilent 2100B Bioanalyzer (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.ĭNA libraries from depleted RNA were generated with the use of the "NEBNEXT Ultradirectional RNA Library Prep Kit" (New England Biolabs) and following the instructions of the manufacturer. RNA concentrations were determined by measuring absorbance at 260 nm. Total RNA was isolated from exponentially growing cells by the “mechanical disruption protocol” using the RNeasy kit (Qiagen, Hilden, Germany), following the instructions of the manufacturer. Saccharomyces cerevisiae strain BY4741 was grown overnight and dilluted to 0.2 OD, allowed to grow until 0.5 and then was grown for 2 hours Saccharomyces cerevisiae strain BY4741 was grown overnight and dilluted to 0.2 OD, allowed to grow until 0.5 and then was grown with poacid acid for 1 hour Reference gene database is refseq_GRCm38.p5.GEO help: Mouse over screen elements for information. Raw files were analyzed using CLC genomics workbench RNA-Seq analysis tools (Qiagen Bioinformatics) The Fastq sequences were aligned and quantitated using the CLC genomics workbench RNA-Seq analysis tools (Qiagen Bioinformatics). Approximately 48,710 reads were obtained per library. Nextera adapters were added and the libraries were pooled and sequenced on an Illumina Hiseq 4000. 21 cycles of pre-amplification were found to be optimal. The TSO, Oligo-dT30VN, and ISPCR primers were biotinylated on the 5’ end. Bulk RNA sequencing was carried out on 0.25ng of RNA in triplicates from each treatment type using the protocol as previously described (Picelli et al., 2014) with the following modifications. RNA was extracted from sorted Live CD45+CD3+CD8+Spas-1+Spas-2- T cells using an Ambion micro RNA isolation kit (AM1931) according to the manufacturer’s protocol. Cells from the five mice from each treatment arm were pooled and split into triplicates. Live CD45+CD3+CD8+Spas-1+Spas-2- T cells were sorted from draining lymph nodes from TRAMP-C2 bearing mice on day 28 after treatment. SP-2-TAGGACC_S2_L001_R1_001: anti-PD-1 DANA_2Īfter tumor injection, mice were treated with their respective checkpoint inhibitor treatments on days 3, 6, and 9 GEO help: Mouse over screen elements for information.
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