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Sample GSM4456927

Query DataSets for GSM4456927

Status

Public on Mar 11, 2021

Title

WG17019_G03-W03

Sample type

SRA

Source name

dorsal root ganglion

Organism

Macaca mulatta

Characteristics

tissue: dorsal root ganglion
cell type: PEP1
gender: Female
age: 4 years 8 months

Extracted molecule

total RNA

Extraction protocol

Extracted DRGs were cut once longitudinally (along the nerve) followed by chopping into ~0.5 mm slices. Samples were enzymatically treated for 1 h at 37°C with triturating every 15 min. The resulting cell suspension was then run through 100 µm cell-strainers. 1 ml of 10% Optiprep was loaded under the cell suspension solution using a gel loading tip and centrifugated at 200g for 6 min with a low break. Supernatant was discarded, the cell pellet resuspended and run through a 10 µm strainer. The strainer was rinsed twice and cells were then collected by flushing the filter twice. The cells were kept on ice for 15-20 min, centrifuged for 3 min at 100 g and resuspended in 900 µl of filtered NMDG-SC/B27/12% optiprep and dispensed immediately into a WaferGen9600 Chip.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

single cell
GTCGTTGAACTGT

Data processing

Reads flagged as invalid by the Illumina HiSeq control software were discarded. In the remaining reads, any 3′ bases with a quality score of B were removed. If any of the six UMI bases in the 5' end had a Phred score <17 the read was discarded, else the UMI was cut away and saved. The following three bases had to be G for the read to be kept. These, as well as any additional up to totally nine (presumably template-switch derived) G:s were cut away. Reads were discarded if the remaining transcript-derived sequence ended in a poly(A) sequence leaving <25 bases, or if the remaining sequence consisted of fewer than six non-A bases or a dinucleotide repeat with fewer than six other bases at either end. The reads were sorted by the well, as defined by the combined two index read barcodes.
Alignment was performed using the Bowtie aligner, allowing for up to three mismatches and up to 24 alternative mappings. Reads with no alignments were realigned against an artificial chromosome, containing all possible splice junctions arising from the exons defined by the UCSC transcript models. The coordinates of aligned splice junctions were translated back to the corresponding genomic positions.
For expression level calculation, the exons of each locus with several transcript variants were merged to a combined model representing total expression of the locus. To account for incomplete cap site knowledge, the 5′ ends of all models were extended by 100 bases, but not beyond the 3′ end of any upstream nearby exon of another gene of the same orientation.
The annotation step was performed separately for each well. For each genomic position and strand combination the number of reads in each UMI was counted. Any multiread that mapped to some repeat outside exons was assigned randomly as one of these repeats and did not contribute to the transcript corresponding to the exon. Else, if the multiread mapped to some exon, and not to any repeat outside exons, it was assigned to the exon where it was closest to the transcript model 5′ end. If it had no exon mapping, it was assigned randomly at one of the mappings. The total number of molecules at each mapping position was determined by the number of distinct UMIs observed. Any UMI represented by only a single read was excluded, in order to reduce false molecules due to PCR and sequencing errors. The raw UMI count was corrected for the UMI collision probability.
Genome_build: rheMac8
Supplementary_files_format_and_content: Processed data consists of a UMI count matrix, where rows are geneIDs and columns cellIDs, and an annotation text file where rows are CellIDs and columns the annotations

Submission date

Apr 07, 2020

Last update date

Mar 11, 2021

Contact name

Sten Linnarsson

Organization name

Karolinska Institutet

Department

Medical Biochemistry and Biophysics

Lab

Molecular Neurobiology