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Sample GSM1280092

Query DataSets for GSM1280092

Status

Public on Dec 20, 2013

Title

ESCell_30_TCGGGTG_single

Sample type

SRA

Source name

R1 mouse embryonic stem cells, single

Organism

Mus musculus

Characteristics

cell line: R1
cell type: mouse embryonic stem cells
barcode: TCGGGTG

Growth protocol

R1 mouse embryonic stem cells were grown in ES1 medium on irradiated MEF feeder cells and harvested at confluency. After trypzinisation, the feeder cells were given time to settle in order to obtain a pure R1 ESC suspension. To reduce contamination with dead and dying cells, dead cells were subsequently stained with Red Fixable Dead Cell Stain (Life Technologies) and dead and early-apoptotic cells were depleted with AnnexinV-conjugated microbeads (Miltenyi Biotec). The cells were resuspended at a concentration of 400 cells/μl in ES1 medium with 10% DMSO, aliquoted and frozen at -80°C.

Extracted molecule

total RNA

Extraction protocol

A 30 μL aliquot of ~12000 cells was thawed and 20 μL C1 Suspension Reagent was added (all "C1" reagents were from Fluidigm, Inc.). 5 μL of this mix was loaded according to the manufacturer's protocol on a C1 Single-Cell AutoPrep IFC microfludic chip designed for 10-17 μm cells, and the chip was then processed on a Fluidigm C1 instrument using the "mRNA Seq: Cell Load (1772x/1773x)" script. This captured one cell in each of up to 96 capture chambers, and took approximately 30 minutes. The plate was then transferred to an automated microscope (Nikon TE2000E) and an image was acquired from each site using μManager (http://micro-manager.org), which took less than 15 minutes.
20 μL lysis buffer (0.15% Triton X-100, 1 U/μL TaKaRa RNase inhibitor, 4 μM reverse transcription primer C1-P1-T31, 5% C1 Loading Reagent and 1:50,000 Life Technologies ERCC Spike-In Mix 1), reverse transcription mix (1x SuperScript II First-Strand Buffer supplemented with 3 mM MgCl2, 1.5 mM dNTP, 4 mM DTT, 3.3% C1 Loading Reagent, 1.8 μM template-switching oligo C1-P1-RNA-TSO, 1.5 U/μL TaKaRa RNase inhibitor and 18 U/μL Life Technologies Superscript II reverse transcriptase) and PCR mix (1.1x Clontech Advantage2 PCR buffer, 440 μM dNTP, 530 nM PCR primer C1-P1-PCR-2, 5% C1 Loading Reagent and 2x Advantage2 Polymerase Mix) were added to the designated wells according to the manufacturer's instructions, but using the indicated mixes in place of the corresponding commmercial reagents. The plate was then returned to the Fluidigm C1 and the "mRNA Seq: RT + Amp (1772x/1773x)" script was executed, which took about 8.5 hours and included lysis, reverse transcription and 21 cycles of PCR. When the run finished, the amplified cDNA was harvested in a total of 13 μL C1 Harvesting Reagent and quantified on an Agilent BioAnalyzer. The typical yield was 1 ng/μL. Amplified cDNA was simultaneously fragmented and barcoded by 'tagmentation', i.e. using Tn5 DNA transposase to transfer adapters to the target DNA. 96 different 10x transposome stocks (6.25 μM barcoded adapter C1-TN5-x, 40% glycerol, 6.25 μM Tn5 transposase, where x denotes a well-specific barcode) were prepared, each with a different barcode sequence. 6 μL harvested cDNA was mixed with 5 μL tagmentation buffer (50 mM TAPS-NaOH pH 8.5, 25 mM MgCl2 and 50% DMF), 11.5 μL nuclease-free water and 2.5 μL 10x transposome stock. The mix was incubated for 5 minutes at 55°C then cooled on ice. 100 μL Dynabeads MyOne Streptavidin C1 beads were washed in 2xBWT (10 mM Tris HCl pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) then resuspended in 2 mL 2xBWT. 20 μL beads was added to each well and incubated at room temperature for 5 minutes. All fractions were pooled, the beads were immobilized and the supernatant removed (thus removing all internal fragments, and retaining only the 5'- and 3'-most fragments). The beads were then resuspended in 100 μL TNT (20 mM Tris pH 7.5, 50 mM NaCl, 0.02% Tween), washed in 100 μL Qiagen Qiaquick PB, then twice in 100 μL TNT. The beads were then resuspended in 100 μL restriction mix (1x NEB NEBuffer 4, 0.4 U/μL PvuI-HF enzyme), designed to cleave 3' fragments carrying the PvuI recognition site. The mix was incubated for one hour at 37°C, then washed three times in TNT. Finally, the single-stranded library was eluted by resuspending the beads in 100 μL 100 mM NaOH, incubating for 5 minutes, removing the beads, then adding 100 μL 100 mM HCl and 50 μL Neutralization buffer (200 mM Tris pH 7.5, 0.05% Tween-20). At this point, the typical yield was 1-5 nM single-stranded library. Aliquots from these were run on the HiSeq 2000. Another aliquot was further amplified by 9 cycles of PCR to produce a double-stranded library as described in Islam, S. et al. Nat. Protoc. 7, 813-828 (2012). These were also run on the HiSeq 2000.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

PolyA RNA

Data processing

Read quality filtering and barcode extraction: Every read that was considered valid by the Illumina HiSeq control software was processed and filtered as follows: a) any 3' bases with a quality score of 'B' were removed; b) the well-identifying barcode was extracted from the 5' end of the read; c) if the read ended in a poly(A)-sequence leaving less than 25 transcript-derived bases, the read was discarded; d) the UMI was extracted and if any of the UMI bases had a Phred score less than 17 the read was discarded; e) a maximum of nine template-switching generated Gs were removed from the 5' end of the transcript-derived sequence; f) if the remaining sequence consisted of less than six non-A bases, or a dinucleotide repeat with less than six other bases at either end, the read was discarded.
Alignment: The reads were aligned to the UCSC mm10 genome using the Bowtie aligner, allowing for up to three mismatches and up to 24 alternative mappings for each read. The genome included an artifical chromosome, containing a concatamer of the ERCC control sequences. Any reads with no alignments were re-aligned against another artifical chromosome, containing all possible splice junctions arising from the exons defined by the known transcript variants. Reads mapping within these splice junctions were translated back to the corresponding actual genomic positions. The UCSC transcript models22 were used for the expression level calculation. If a locus had several transcript variants, the exons of these were merged to a combined model that represented all expression from the locus. To account for incompete 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.
Annotation and quantitation: The annotation step was performed barcode-by-barcode. For every unique mapping (genomic position and strand), the number of reads in each UMI was counted. Any multiread that had one or more repeat mappings that was outside exons, was assigned randomly as one of these repeats and contributed to the summarized read count of that repeat class. Else, if it had one or more mappings to exons, it was assigned at the exon where it was closest to the transcript model 5' end, even if the sequence was repeat-like. If it had no exon mapping, it was assigned randomly at one of the mappings. After assigning reads, the number of molecules at each mapping position was determined by the number of distinct UMIs observed. To account for UMIs that stem from PCR-induced mutations or sequencing errors, any UMI that had fewer reads than 1/100 of the average of the non-zero UMIs was excluded. The raw UMI count was corrected for the UMI collision probability (important only at high UMI counts) as described. The expression level of each transcript model was taken as the total number of molecules at all its possible mapping positions.
Genome_build: GRCm38
Supplementary_files_format_and_content: Tab-delimited text file containing counts of detected cDNA molecules for each transcript model in each of the 96 cells, using data from all three sequencing lanes.

Submission date

Dec 04, 2013

Last update date

May 15, 2019

Contact name

Sten Linnarsson

E-mail(s)

[email protected]

Phone

+46852487577

URL

http://ki.se