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Sample GSM4060158

Query DataSets for GSM4060158

Status

Public on Dec 31, 2019

Title

SAM19

Sample type

SRA

Source name

RNA extracted from PBMCs after stimulation with F1/V vaccine antigen for 24 hours

Organism

Homo sapiens

Characteristics

subject: SUBA
time: Day 14
treatment: Flagellin/F1/V 10 mug
cell type: peripheral blood mononuclear cells (PBMCs)

Treatment protocol

Stimulated PBMC with F1/V vaccine antigen for 24h. Wash once with PBS. Then transfer 5e6 – 1e7 cells into a 1.5ml snap top tube and centrifuge cells at 2000g for 5 minutes at room temperature. Remove supernatant completely, leaving cell pellet intact. Put cell pellet on ice and take samples to a dedicated RNA extraction laboratory.

Extracted molecule

polyA RNA

Extraction protocol

Add 600ul Buffer RLT Plus (with 2ME) and vortex for 30 seconds. Transfer lysed cells to a gDNA Eliminator spin column that is sitting in a 2ml collection tube that is supplied Centrifuge at 11,000 rpm for 30 seconds. Discard the column and save the flow-through. Add 600ul of 70% ethanol to the flow-through and mix by pipetting. Transfer half of the sample into an RNeasy spin column which has a 2ml collection tube supplied. Close lid and centrifuge for 15 seconds at 11,000 rpm. Discard the flow-through into the waste bag on the bench and place the column onto the 2ml tube again. Transfer the other half of the sample into the RNeasy spin column and centrifuge once again for 15 seconds at 11,000 rpm. Add 700ul Buffer RW1 to the RNeasy spin column. Close lid and centrifuge for 15 seconds at 11,000 rpm and then discard the flow through. Add 500ul Buffer RPE to the RNeasy spin column. Close the lid and centrifuge for 15 seconds for 11,000 rpm and then discard the flow through. Add 500ul Buffer RPE to the RNeasy spin column again. Close lid and centrifuge for 2 minutes at 11,000 rpm. Remove spin column gently out of the centrifuge to avoid any splashing of buffer onto the column. Discard the flow through and centrifuge RNeasy spin column (without any buffer) for 1 min at 14,000 rpm (full speed) to further dry the membrane. Place the RNeasy spin column into a new 1.5ml collection tube (supplied with kit) and add 50ul of RNase-free water (supplied with kit) directly onto the middle of the spin column membrane (without touching membrane with tip).Close lid and centrifuge for 1 minute at 11,000 rpm to elute the RNA. Place RNA on ice and discard the RNeasy spin column.
Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 1 to 3ug of total RNA. Ribosomal RNA was removed by a hybridization method using Ribo-ZERO kits (Illumina-EpiCentre). mRNA was then fragmented in buffer containing 40mM Tris Acetate pH 8.2, 100mM Potassium Acetate and 30mM Magnesium Acetate and heating to 94 degrees for 150 seconds. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3’ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-3000, using single reads extending 50 bases.
Fragments were sequenced on an Illumina HiSeq-3000, using single reads extending 50 bases. Fourty-one libraries were multiplexed across 4 sequencing lanes with targeted coverage of 39 million reads per RNA-Seq library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Data processing

The latest version of the human reference genome assembly (GRCh38), gene models, and associated gene annotation information in the form of a Gene Transfer Format (GTF) file were obtained from the ENSEMBL database (Version 90). X and Y chromosomes were excluded to avoid gender-specific effects.
Sequences of known human rRNAs, rRNA pseudogenes, tRNA pseudogenes, mitochondrial rRNAs (mt-rRNAs), mitochondrial tRNAs (mt-tRNAs), and mt-rRNA pseudogenes were obtained from the Ensembl database (Version 90, August 2017) using the biomaRt R package (Version 2.32.1). Known human tRNA sequences were obtained from the Ensembl database (Version 90, August 2017) using the Ensembl Perl API (Version 90). These sequences were then used to build a Bowtie2 index of contaminant sequences.
Adapter sequences and low-quality 5’ ending sequences were removed from raw sequencing reads using the Trimmomatic software (Version 0.36).
The impact of quality filtering was assessed using the FastQC software.
Following adapter removal, sequence reads were aligned to the index of known human rRNAs and tRNAs using Bowtie2 with its local alignment option. Reads that mapped to the index were removed, and those that did not were output to a FASTQ file for alignment to the reference genome.
Filtered reads were aligned to the reference transcriptome/genome using the HISAT2 splice-aware sequence aligner (Version 2.1.0)
For each sample, the quality of reference alignments was evaluated using the RSeQC software (Version 2.6.4)
Quantification was carried out on the gene level using the featureCounts function of the Subread software package (Version 1.5.3). Reads that overlapped with multiple genes or mapped to multiple locations on the reference genome were excluded. Known ribosomal, transfer, and mitochondrial RNA genes were removed from the final read count analysis dataset
Genome_build: GRCh38
Supplementary_files_format_and_content: abate_npj_2019_rnaseq_featurecounts.tab.gz contains read counts as assigned by Subread featureCounts (Version 1.5.3). Rows represent genes, and columns represent samples.

Submission date

Sep 04, 2019

Last update date

Dec 31, 2019

Contact name

Johannes Goll

Organization name

The Emmes Company

Department

Bioinformatics Service Group

Street address

401 N Washington St