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Status |
Public on Dec 19, 2016 |
Title |
PRO20150626_BatchA_replicate2 |
Sample type |
SRA |
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Source name |
HEK293
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Organism |
Homo sapiens |
Characteristics |
cell line: HEK293 batch: A
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Growth protocol |
HEK293 cells were cultured in DMEM medium supplemented with glutamine and 10% fetal calf serum.
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Extracted molecule |
total RNA |
Extraction protocol |
1000 HEK-293 cells in 4.5 ul C1 wash buffer (Fluidigm) were lysed with 9 ul of lysis buffer (0.2% w/v Tween 20, 1 U/ul Promega RNAsin RNAse inhibitor, 2 µM reverse transcription primer (TGCGGTATCTAAAGCGGTGAGT(30)VN), 2.5 mM dNTPs, 1x C1 loading reagent (Fluidigm), ERCC Spike-In Mix 1 @ 20,000 molecules/cell (Life Technologies). The sample was incubated for 10 min at room temperature followed by 3 min at 70°C and 3 min at 10°C. Reverse transcription: 18 ul of 1.75 x reverse transcription mix, 8.75 mM DTT, 1.75 M Betaine, 10.5 mM MgCl2, 1.75 uM template switching oligonucleotide (GCAATGAAGTCGCAGGGTTGN(4)H(4)rGrGrG), 0.5 U/ul Promega RNAsin, 5.5 U/ul Life Technologies Superscript II reverse transcriptase, 1x C1 (Fluidigm) loading reagent were added to the lysed cells and the sample was incubated for 10 min at 25°C, 90 min at 42°C, 15 min at 70°C and kept below 10°C until PCR amplification. PCR amplification and barcoding: One tenth of the reverse transcription (3.15 µl, 100 cells) was mixed with 27 µl 1.15 x Kappa KAPAHiFiHiFi HotStart Ready Mix, 55 nM barcode primer (TGCGGTATCTAAAGCGGTGAGCCATCTCATCCCTGCGTGTCTCCGACTCAG<index>GCAATGAAGTCGCAGGGTTG, indices are Ion Torrent standard indices), 1.1 µM biotinylated PCR primer (Bio-TGCGGTATCTAAAGCGGTGAG). For PCR amplification samples were incubated 3 min at 98°C followed by 18 cycles at 98°C for 20 sec, 64°C for 15 sec, 72°C for 6 min, and a final extension at 72°C for 5 min. Fragmentation and library preparation: PCR products were fragmented by tagmentation with EzTn5 transposase (Epicentre) and Tn5 mosaic end duplexes (upper: 5’-AGA TGT GTA TAA GAG ACA-3’, lower: 5’-PhosCTG TCT CTT ATA CAC ATC T-3’), 5’ terminal (biotinylated) fragments were isolated with streptavidin beads and and Ion Torrent adapter sequences were added by PCR (Primers: CCATCTCATCCCTGCGTGTCTC, 0.5 μM; CCACTACGCCTCCGCTTTCCTC, 0.5 μM; CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATAGATGTGTATAAGAGACAG, 0.125 μM). 5' selective, oligo-dT-primed RNA-seq based on the SmartSeq technique.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Ion Torrent Proton |
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Description |
PRO20150626_0207_dge_ed1.txt
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Data processing |
Cutadapt-1.2.1 of 3p adaptor 'CTGTCTCTTATACACATCT', Discarding of trimmed reads with length < 50b, Filtering of reads not starting with a perfect 14b 5p adaptor 'AAGTCGCAGGGTTG', then a well formatted HUMI sequence (N4H4) followed by at least 3 'G' before template sequence, Extraction of 5p adaptor, UMI and strech of 'G', Mapping with STAR_2.4.0a versus hg19, Drop-seq_tools-1.0 dropseq.jar tool for tagging with Gene information from Ensembl release GRCh37.75, then DigitalExpression calling with default parameters (edit distance=1). Genome_build: hg19 Supplementary_files_format_and_content: PRO20150626_0207_dge_ed1.txt: quantifcation file containing for each gene detected in at least one sample the number of mRNA molecules counted for this gene.
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Submission date |
Mar 11, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Kevin Lebrigand |
Organization name |
IPMC/CNRS
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Lab |
Functional Genomics Platform of Nice-Sophia-Antipolis, France.
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Street address |
660 route des lucioles
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City |
Valbonne - Sophia-Antipolis |
ZIP/Postal code |
06560 |
Country |
France |
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Platform ID |
GPL17303 |
Series (2) |
GSE79123 |
HEK293 cells 100 cell RNAseq profiling on Ion Proton |
GSE79136 |
A cost effective single cell RNA sequencing approach for the Fluidigm C1 |
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Relations |
BioSample |
SAMN04546560 |
SRA |
SRX1629285 |
Supplementary data files not provided |
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Raw data are available in SRA |
Processed data are available on Series record |
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