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Sample GSM1819051

Query DataSets for GSM1819051

Status

Public on Jul 14, 2015

Title

TRA00028928_ls_13

Sample type

SRA

Source name

thymus

Organism

Mus musculus

Characteristics

cell type: MEC
mec_type: WT_GFPhi
strain: B6xNOD
Sex: Female
age: 5 week-old
sorting markers: CD45-,Ly51-,MHCII+,GFPhi
primer: ls_13
genotype/variation: WT; AireGFP

Growth protocol

Sorted live cells from thymi of Aire wildtype or KO mice. All mice carried Aire-GFP transgene as marker for Aire expression.

Extracted molecule

total RNA

Extraction protocol

Thymi were removed and digested in collagenase/dispase to single cell suspensions. Medullary thymic epithelial cells were sorted by FACS as CD45-Ly51-MHCII+GFP+. Single cells were sorted into water in wells of 96well plate and lysed by freeze-thaw.
mRNA was reverse transcribed using adapters containing oligo dT and ArrayScript (Ambion), second strand synthesis was performed using Second strand synthesis module (NEBNext #E6111L), and linear amplification was performed by in vitro transcription using MEGAshortscript T7 transcription kit (Ambion AM1354). Illumina RNA adapters were ligated onto transcripts using T4 RNA Ligase 2, truncated (Enzymatics L6070L) and processed according to standard Illumina protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

B6xNOD;AireGFP

Data processing

demultiplex by cell barcode (custom scripts)
tophat2 alignment
Details:
Raw data were processed using custom scripts. Read 1 contains the transcript sequence, and Read 2 contains the single cell barcode and unique molecular identifiers. Raw reads were first trimmed using the FASTX-Tollkit v0.0.13 (fastx_trimmer –Q 33). Read 2 was trimmed in order to extract the single cell barcode (8bp) and the UMI (4-8bp), and Read 1 was trimmed to 30bp get rid of a potential oligo-dT sequence. After merging the different parts (barcode, umi and transcript sequence), reads were filtered for quality (more than 80% of the sequence having a Sanger Phred+33 quality score > 33) using fastq_quality_filter -v -Q 33 -q 20 -p 80. Then the reads were assigned to each single cell by using the 8bp barcode and the fastx_barcode_splitter.pl tool script for a maximum of 2 mismatches. Reads assigned to each single cell were then trimmed again to retrieve the transcript sequence using fastx_trimmer.
Mapping was performed using Tophat2 to the mm10 mouse transcriptome and keeping the strand information with the following options : tophat -p 2 --library-type fr-firststrand --read-mismatches 5 --read-gap-length 5 --read-edit-dist 5 --no-coverage-search --segment-length 15 --transcriptome-index. Duplicated mapping reads were filtered out using the unique molecular barcodes as follows. First duplicated mapped reads were marked using picard-tools-1.79/MarkDuplicates.jar. Then the genomic position of the duplicated reads were extracted and for each of these positions, only reads having unique molecular identifiers were then kept. Reads that mapped to multiple positions were filtered out using samtools 0.1.19 flag 256. Finally reads were assigned to genes using htseq-count, biomart_mm10_gene.gff and the following options : -s yes -m intersection-nonempty. The script was modified in order to assign reads that overlapped in several genes to the one closest to a 3’ end.
Genome_build: mm10
Supplementary_files_format_and_content: SCS_MEC.csv: Raw read counts.
Supplementary_files_format_and_content: SCS_MEC_metadata_table.txt: Metadata, including genotype information for the single cells.

Submission date

Jul 11, 2015

Last update date

May 15, 2019

Contact name

CBDM Lab

E-mail(s)

cbdm@hms.harvard.edu

Phone

617-432-7747

Organization name

Harvard Medical School