 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 22, 2018 |
| Title |
WG17009_C04-W13 |
| Sample type |
SRA |
| |
|
| Source name |
striatum
|
| Organism |
Mus musculus |
| Characteristics |
strain: 5ht3a-EGFP
Sex: male
postnatal days: 63
cell type: Migrating
|
| Growth protocol |
Mice were housed on a standard 12+12 light/dark cycle with 2-5 mice per cage
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Dorso-lateral striatum (bregma, AP: 1.42 to -0.58 mm) from Lhx6Cre:RFP, Pvcre:TdT and Htr3aEGFP mice was dissociated into a single cell suspension as described in Zeisel et al., Science vol. 347 p.1138 (2015) . Dissociated cells were FACS sorted based on fluorescence (RFP+ and EGFP+) and positive cells were sorted using BD FACSAria II SORP into a 9600 multiwell array (2400 cells/plate) prefilled with 50 nl lysis buffer (500nM STRT-P1-T31, 4.5nM dNTP, 2% Triton-X-100, 20mM DTT, 1.5U/μl TaKaRa RNase Inhibitor), followed by 3min lysis at 72°C.
85nl reverse transcription (RT) mix (2.1X SuperScript II First-Strand Buffer, 12.6mM MgCl2, 1.79M betaine, 14.7U/μl SuperScript II, 1.58U/μl TaKaRa RNase Inhibitor, 10.5μM P1B-UMI-RNA-TSO) were dispensed and RT carried out 42°C for 90 minutes. 96 Transposome stocks were assembled (6.25μM barcoded adapter (STRT-Tn5-Idx[1-96]), 6.25μM Tn5 transposase, 40% glycerol), 37°C 1h. Tn5 reactions were assembled with 3μl transposome and 2μl amplified cDNA, in a total 20μl 1x CutSmart buffer (NEB), and incubated at 55°C 20min.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| 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. For the human samples all reads mapping anywhere within the whole locus, defined as the region (including actual introns) from the start of the 100 base 5'end extension of the first exon up to the end of the last exon, were counted as exon-derived.
Genome_build: UCSC mm10
Supplementary_files_format_and_content: Tab-delimited table of total number of detected mRNA molecules from each gene in each cell
|
| |
|
| Submission date |
Nov 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Sten Linnarsson |
| Organization name |
Karolinska Institutet
|
| Department |
Medical Biochemistry and Biophysics
|
| Lab |
Molecular Neurobiology
|
| Street address |
Scheeles väg 1
|
| City |
Stockholm |
| ZIP/Postal code |
171 65 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE106707 |
Single cell RNA sequencing of interneurons of the mouse dorsolateral striatum II |
| GSE106708 |
Single cell RNA sequencing of interneurons of the mouse dorsolateral striatum |
|
| Relations |
| BioSample |
SAMN08001189 |
| SRA |
SRX3376233 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
