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Sample GSM2983935 Query DataSets for GSM2983935
Status Public on Sep 10, 2019
Title L15312_NC-wo_05_D06
Sample type SRA
 
Source name negative control (no source)
Organism synthetic construct
Characteristics strain: negative control (no strain)
tissue: n/a
genotype: n/a
age: n/a
Extracted molecule total RNA
Extraction protocol To isolate Aldh1b1 expressing cells and their progeny, 1 mg of corn oil dissolved tamoxifen (20 mg/ml; Sigma) was injected intraperitoneally in Aldh1b1CreERT2 / ROSA26 LSLtdTomato double heterozygous adult mice for 5 consecutive days. Aldh1b1 expressing cells and their early progeny were isolated one day later from pancreata dissociated into single cells by virtue of their tdTomato (PE-Texas Red channel) expression from the lineage tracing mouse pancreas by FACS. 
Pancreata were dissociated into single cells and cells were FACsorted into a 96 well plate containing 2 ul of nuclease free water with 0.2% Triton-X 100 and 4 U murine RNase Inhibitor (NEB), spun down and frozen at -80°C. Lysis, reverse transcription of the RNA and cDNA amplification was performed according to the smartseq-2 protocol 6 with few modifications as detailed below. After thawing the samples, 2 ul of a primer mix (5 mM dNTP (Invitrogen), 0.5 uM dT-primer*, 4 U RNase Inhibitor (NEB)) was added. After filling up to 10 ul with RT buffer mix for a final concentration of 1x superscript II buffer (Invitrogen), 1 M betaine, 5 mM DTT, 6 mM MgCl2, 1 uM TSO-primer*, 9 U RNase Inhibitor and 90 U Superscript II, RNA was denatured for 3 minutes at 72°C and reverse transcription was performed at 42°C for 90 min. The reverse transcriptase was then inactivated at 70°C for 15 min. The cDNA was amplified using Kapa HiFi HotStart Readymix (Peqlab) at a final 1x concentration and 0.1 uM UP primer* under the following cycling conditions: initial denaturation at 98°C for 3 min, 22 cycles [98°C 20 sec, 67°C 15 sec, 72°C 6 min] and final elongation at 72°C for 5 min. The amplified cDNA was purified using 1x volume of hydrophobic Sera-Mag SpeedBeads (GE Healthcare) and the DNA was eluted in 12 ul nuclease free water. The concentration of the samples was measured with a Tecan plate reader Infinite 200 pro in 384 well black flat bottom low volume plates (Corning) using AccuBlue Broad range chemistry (Biotium). For library preparation 700 pg cDNA in 2 ul was mixed with 0.5 ul tagment DNA enzyme and 2.5 ul tagment DNA buffer (Nextera, Illumina) and tagmented at 55°C for 5 min. Subsequently, Illumina indices were added during PCR (72°C 3 min, 98°C 30 sec, 12 cycles [98°C 10 sec, 63°C 20 sec, 72°C 1 min], 72°C 5 min) with 1x concentrated KAPA Hifi HotStart Ready Mix and 0.7 uM dual indexing primers. After PCR, libraries were quantified with AccuBlue Broad range chemistry, equimolarly pooled and purified twice with 1x volume Sera-Mag SpeedBeads. This was followed by Illumina sequencing on a Nextseq500 aiming at an average sequencing depth of 0.5 mio reads per cell. Primers were as follows: dT primer: Aminolinker-AAGCAGTGGTATCAACGCAGAGTCGACT(30)VN; Template Switch Oligo (TSO) primer: AAGCAGTGGTATCAACGCAGAGTACATrGrGrG; Universal Part (UP) primer: AAGCAGTGGTATCAACGCAGAGT.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description no RNA
Data processing basecalling: bcl2fastq 2.17.1.14
alignment: alignment of single-end data against mm10 with gsnap version 2017-03-17; splice sites were supported by using Ensembl version 87 (gsnap -d mm10 --gunzip --use-sarray=1 -B 4 -t 16 -N 1 -s EnsemblGene-87.ss.mm10.iit -n 1 -A sam)
read counting: counting was done with featureCounts version 1.5.2 (-a EnsemblGene-87.mm10.TR.gtf -s 2 -Q 1) and the Ensembl version 87 gtf file
quality filtering: qc was done with the scater package and cells removed based on various quality metrics (ERCC content, chrM, total Features, total Counts and cell cycle)
Genome_build: mm10
Supplementary_files_format_and_content: tab delimited file from featureCounts (featureCounts.mm10.e87.tsv.gz); columns are in the following order: Ensembl Gene ID, Chromosom, Exon Start Coordinates, Exon End Coordinates, Gene Length; Counts from the sequenced single cell libraries
Supplementary_files_format_and_content: tab delimited file (scater.cpmlog10_counts.tsv.gz) created with the scater package; columns are the remaining cells and rows are genes
 
Submission date Feb 07, 2018
Last update date Sep 10, 2019
Contact name Anthony Gavalas
Organization name TU Dresden
Street address Fetscherstr. 105
City Dresden
ZIP/Postal code 01307
Country Germany
 
Platform ID GPL19604
Series (1)
GSE110283 Single cell RNA-Seq of adult mouse pancreatic stem cells and early progenitors
Relations
BioSample SAMN08475935
SRA SRX3657488

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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