title: 1772122305_F02

description: ESMoleculeCounts.cef


title: 1772122305_B10

status: Public on Oct 06 2016

type: SRA

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

description: ESMoleculeCounts.cef

library_source: transcriptomic

data_processing:
Read processing was performed as described (Islam et al., Nat Methods. 2014 Feb;11(2):163-6), except that we removed any RNA molecule (i.e. Unique Molecular Identifier) supported only by a single read ("singleton molecules"). This removed a large number of false positive molecules, artefacts that can arise by sequencing error, PCR-induced mutations or translocations and cross-contamination.
The first 6 bases of each read represent the random Unique Molecular Identifier used for molecule counting. After follows three or more Gs, stemming from the template switching at the mRNA 5' end during first strand cDNA synthesis. In some reads, one of the three Gs has been replaced by an A by the Illumina HiSeq software, representing failed base calling at that position. We rejected cells that had less than 2000 mRNA molecules, more than 26,000 mRNA molecules (putative doublets) as well as cells that formed a separate cluster with no specific gene expression (putative damaged or dead cells). Genes that were detected at less than 4 molecules in the whole datasets were eliminated.
Genome_build: UCSC mm10 and UCSC hg19
Supplementary_files_format_and_content: Tab-delimited table of total number of detected mRNA molecules from each gene in each cell identified by the "Title" field in Samples list. Mitochondrial genes and genes with less than 4 molecules have been omitted. The first two lines of each file are table metadata (see https://github.com/linnarsson-lab/ceftools)

molecule: polyA RNA

organism: Homo sapiens

source_name: stem cells

growth_protocol: Wild-type CD1 mice were mated overnight and noon of the day was considered E0.5. Mothers were killed and embryos were dissected out of the uterine horn between the time points E11.5 and E18.5. Adult CD1 animals were dissected at P19 to P27. Human tissue was collected at Addenbrooke’s Hospital (Cambridge, UK) and dissected in HIBERNATE medium (Gibco). Samples were shipped overnight on ice (in HIBERNATE solution) to Sweden. For the embryonic stem cells (hES DA),  iCell DopaNeurons from Cellular Dynamics International (DNC-301030001, lot #6003358) were differentiated following the manufacturer’s recommendations and harvested at day 5 and 21.

extract_protocol:
Human embryo ventral midbrain dissection was performed on ice in neuronal N2 culturing media. 10-17  tissue pieces were collected for each experiment. The tissue pieces were dissociated using the Papain dissociation system (Worthington) following manufacturers recommendations, adjusting incubation time based on tissue piece size to 25-45 mins. Using glass pipettes of increasingly smaller tip diameter, the cell suspension was then filtered with 20um filter (Partec) and cells kept in N2 media, and cells loaded into C1 chips for cell capture.
Fluidigm C1 Autoprep System  Cells medium (10-17 micrometer) microfluidic was used to capture the cells. 14 microlitre of cell suspension (approx. 800 cells/microlitre in N2 culturing medium with DNaseI) was mixed with 7 microlitre C1 Suspension Reagent after filtering. Single-cells were then captured for 30 min at 4°C using the “Cell Load (1772x/1773x)” script. The protocol for Lysis, RT and PCR was performed as previously described  (Islam et al., Nat Methods. 2014 Feb;11(2):163-6). Amplified cDNA was harvested with 13 microlitre Harvest Reagent and cDNA library quality was measured on an Agilent BioAnalyzer. Cell barcoding and fragmentation was performed in a single step using Tn5 DNA transposase as described previously. 1 microlitre Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were resuspended in 20 microlitre Binding and Blocking buffer (10mM Tris, 250mM NaCl, 5mM EDTA, 0.5% SDS) and added to each well. After 15 min incubation at room temperature, all wells were pooled, the beads washed once with 100 microlitre Washing buffer (10mM Tris-150mM NaCl, 0.02% Tween), once in 100 microlitre Qiagen Qiaquick PB and then twice using 100 microlitre Washing buffer. Restriction was performed to cleave 3’ fragments: the beads were incubated in 100 microlitre restriction mix (1x NEB CutSmart, 0.4 U/microlitre PvuI-HF enzyme) for 1h at 37°C. Finally, the beads were washed three times with Washing buffer, then resuspended in 30 μl ddH2O and incubated for 10 min at 70°C to elute the DNA. AMPure beads XP (Beckman Coulter) were used at 1.8x volume and eluted in 30 microlitre to remove short fragments.

tag: tissue

value: stem cells

tag: Sex

tag: age

tag: inferred cell type


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