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Sample GSM7104280

Query DataSets for GSM7104280

Status

Public on Jan 28, 2024

Title

atn_sample1

Sample type

SRA

Source name

Anterior thalamic nuclei

Organism

Mus musculus

Characteristics

genotype: WT
tissue: Anterior thalamic nuclei

Extracted molecule

total RNA

Extraction protocol

Microdissect, dissociate, and manually harvest cells across acute brain slices in B6 mice.
Cells were placed into 8-well strips containing cell collection buffer, and each strip of cells was flash frozen on dry ice, then stored at -80°C until cDNA synthesis. For cDNA synthesis, cells were lysed by adding 1 µL lysis mix and 1 µL 10 µM barcoded RT primer. The samples were incubated for 5 minutes at 50°C to lyse the cells, followed by 20 minutes at 80°C to inactivate the Proteinase K. Reverse transcription master mix was added to the lysis reaction and incubated at 42°C for 1.5 hour, followed by 10 minutes at 75°C to inactivate reverse transcriptase. PCR was performed, and samples were pooled across the plate to yield approximately 2 mL pooled PCR reaction. From this, 500 µL was purified, washed twice with 75% ethanol, and eluted in 20 µL nuclease-free water.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Data processing

Trim input file and remove adapters.
Tag reads with cell barcode and UMI
Align reads to PhiX, then to mouse.
Aggregate reads and de-duplicate UMIs.
Post-processing in R with custom scripts and Seurat.
Assembly: NCBIM37
Supplementary files format and content: Tab delimited file containing count values.

Submission date

Mar 17, 2023

Last update date

Jan 28, 2024

Contact name

Mark Steven Cembrowski

Organization name

University of British Columbia

Department

Cellular and Physiological Sciences

Lab

Cembrowski