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Sample GSM2706907 Query DataSets for GSM2706907
Status Public on Dec 06, 2017
Title WG960011_F08-W06
Sample type SRA
 
Source name somatosensory cortex
Organism Mus musculus
Characteristics cluster: L4_Kcnab3
tissue: somatosensory cortex
strain/background: CD-1
Growth protocol Male and female wild type CD-1 mice (Charles River) between postnatal days 21-37 were used. Postmortem human brain tissue was provided to the Allen Institute for Brain Science by the San Diego Medical Examiner’s (SDME) office.
Extracted molecule total RNA
Extraction protocol Mice were anesthetized with isoflurane, perfused with ice-cold aCSF, brains collected and sectioned using a vibratome or brain matrix and the somatosensory cortex was microdissected. Single cell suspensions were generated using the Worthington Papain dissociation system. Human nuclei were isolated from a -80°C frozen tissue piece using Dounce homogenization.
50nl lysis mix (500nM STRT-P1-T31, 4.5nM dNTP, 2% Triton-X-100, 20mM DTT, 1.5U/μl TaKaRa RNase Inhibitor) was dispensed, followed by 3min lysis at 72°C. Then, 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 2000
 
Description Processed data file: molecules_mouse.xlsx
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 (GRCm38) and hg19 (GRCh37)
Supplementary_files_format_and_content: *xlsx: Excel tables of mRNA molecule counts with genes in rows and cells in columns.
 
Submission date Jul 18, 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 GPL13112
Series (1)
GSE101601 A novel addressable 9600-microwell array single cell RNA-seq method applied on fresh mouse cortical cells and frozen human cortical nuclei
Relations
BioSample SAMN07363732
SRA SRX3016190

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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