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Sample GSM4768530

Query DataSets for GSM4768530

Status

Public on Sep 05, 2020

Title

poolA-31_S127

Sample type

SRA

Source name

Csf1r lineage endothelial cell (CLEC)

Organism

Mus musculus

Characteristics

facs gating: CD45- CD31+ GFP+
tissue: lymph node

Extracted molecule

total RNA

Extraction protocol

Single cells were sorted (BD FACSAriaIII) in 96 well plates (VWR) containing 2 μL of PBS including 0.2% Triton X-100 and 4U of RNase inhibitor (Takara) per well. Plates were properly sealed and spun down at 2000 g for 1 min before storing at −80°C. Whole transcriptome amplification was performed with a modified SMART-seq2 protocol as described previously, using 20 instead of 18 cycles of cDNA amplification. PCR purification was realized with a 0.8:1 ratio (ampureXP beads:DNA). Amplified cDNA quality was monitored with a high sensitivity DNA chip (Agilent) using the Bioanalyzer (Agilent)
Sequencing libraries were performed using the Nextera XT kit (Illumina) as described previously, using 1/4th of the recommended reagent volumes and 1/5th of input DNA with a tagmentation time of 9 min. Library quality was monitored with a high sensitivity DNA chip (Agilent) using the Bioanalyzer (Agilent). Indexing was performed with the Nextera XT index Kit V2 (A-D).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

BAM files were converted to merged, demultiplexed FASTQ files, cleaned using fastq-mcf (ea-utils r819), and QC checked with FastQC (0.11.4). Reads were then mapped to the mouse genome (mm18) using STAR (2.4.1b) and quantified with Subread (1.4.6-p2). Cells with less than 100,000 reads and/or 1000 genes expressed, more than 20% mitochondrial reads, and less than an average expression level of 3.0 of about 80 housekeeping genes 61 were discarded. 469 cells passed these stringent quality criteria
Genome_build: mm10
Supplementary_files_format_and_content: txt

Submission date

Sep 04, 2020

Last update date

Jun 04, 2021

Contact name

Florian Rambow

E-mail(s)

[email protected]

Organization name

VIB

Street address

Herestraat 49 box 912