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Sample GSM2983933

Query DataSets for GSM2983933

Status

Public on Sep 10, 2019

Title

L14385_NC-wo_03_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