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Sample GSM5358990

Query DataSets for GSM5358990

Status

Public on Jun 05, 2021

Title

SSF-1781_A2_CTRL-

Sample type

SRA

Source name

Water

Organism

blank sample

Characteristics

cell type: None
plate: SSF-1781
plate_location: A2
pid: NA
stimulation: NA
visit: NA
site: NA
age: NA
vaccine: NA
case: NA
match: NA
sid: NA
date_m0: NA
date_m3_5: NA
time_to_malaria: NA
malaria_status: NA
waz: NA
haz: NA
hb: NA
Sex: NA
age_weeks: NA
malaria_before_m3_5: NA
malaria_transmission: NA

Extracted molecule

polyA RNA

Extraction protocol

RNA was extracted at the Center for Infectious Disease Research (CIDR, Seattle) using the Promega SV 96 RNA Isolation kit following the manufacturer’s protocol. Samples kept in RNAprotect were centrifuged at 4,000g for 7 minutes at 4°C, cell pellets were resuspended in 150 μL RLT Buffer, and 150 μL of 70% ethanol was added prior to processing with Promega SV 96 RNA Isolation kit. RNAs were eluted with 100 μL nuclease free water. Each 96-well extraction batch was spot checked by Bioanalyzer using Agilent RNA 6000 Pico chip and had an average RIN score of 7.4. RNA samples were distributed in 384 well plates for library preparation. Samples from the same individuals were in the same plate and key study variables (vaccine, site and cases-controls) were checked for balance across plates to avoid batch effects.
An optimized version of Digital Gene Expression (DGE) was used, further reducing the reverse transcriptase reaction volume. In brief, poly(A)+ mRNA from antigen-stimulated PBMCs was linked to unique molecular identifiers (UMIs) using a template-switching reverse transcriptase. Then, cDNA from multiple cells were pooled, amplified, and prepped for multiplexed sequencing using a transposon-based fragmentation method, enriching for 3′ ends and preserving strand information. Nextera XT library preparation and sequence paired-ends were employed on an Illumina NextSeq instrument at the Broad Institute.
SCRB-Seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

reads were aligned using BWA Aln version 0.7.10 using UCSC RefSeq (Human 19) with mitochondrial genes added
Quantified samples were then quality controlled using mapping summary statistics to remove low quality samples based on predetermined minimum values for the total number of mapped reads, percent of mapped reads mapped to the human genome, etc.
Downstream analysis was applied only to reads that mapped uniquely to a UMI and only mapped to isoforms of the same gene (UMI.unq).
The TMM normalization method was applied to account for differing number of read counts and to address unwanted technical variation. The voom transformation from the limma R package was applied to transform the data into a form appropriate for linear models.
Genome_build: hg19
Supplementary_files_format_and_content: concatenated_unique_refseq_umi_counts.tsv contains the count matrix for all samples/cells

Submission date

Jun 04, 2021

Last update date

Jun 06, 2021

Contact name

Carlota Dobaño

Organization name

ISGlobal, Hospital Clínic - Universitat de Barcelona

Street address

Rosselló 153

City

Barcelona