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| Status |
Public on Feb 12, 2018 |
| Title |
C1RamDA_PrE_06 |
| Sample type |
SRA |
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| Source name |
PrE_single cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: Primitive endoderm (PE) cell
sample type: C1-captured cell
starting amount: single cell
cell cycle phase: All phase
method subtype: C1-RamDA-seq
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| Treatment protocol |
For the assessment of PrE differentiation, 5G6GR ES cells were cultured in differentiation medium containing 100 mmol/l dexamethasone rather than blasticidin. The cells are cultured for 72 hours.
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| Growth protocol |
5G6GR mouse ES cells were used for bulk and single-cell RNA-seq. The cells were cultured in a feeder-free gelatin-coated dish in 10% fetal calf serum containing Glasgow minimal essential medium (GMEM; Sigma-Aldrich), 1000 U/mL leukemia inhibitory factor (ESGRO; Invitrogen), 100 µmol/l 2-mercaptoethanol (Nacalai Tesque), 1× non-essential amino acids (Life Technologies), 1 mmol/L sodium pyruvate (Life Technologies), 2 mmol/L L-glutamine (Nacalai Tesque), 0.5× penicillin/streptomycin (Life Technologies), 0.5 ug/ml puromycin (Sigma) and 10 µg/mL blasticidin (Life Technologies).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using an RNeasy Mini Kit (Qiagen). For ribosomal RNA depleted total RNA-seq, depletion of rRNA from 1 µg of ES total RNA was performed by using GeneRead rRNA Depletion Kits (Qiagen). For single-cell RamDA-seq, single cells were directly sorted into the cell lysis buffer containing RNasein plus (Promega), Cell Lysis Buffer (Roche), NP40 (Thermo), and RNase free water (TaKaRa). For averaged G1-phase cell lysate sample, 180 cells were sorted into 180 µl of the cell lysis buffer and individually divided into 1 μl in a 96-well PCR plate after well-mixing to lyse the cells. The cell lysate solution was stored at -80ºC until use.
For bulk RNA-seq, the libraries were constructed by using a commercial kit (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; NEB) in accordance with the manufacturer's protocol, with slight modifications during the reverse-transcription and PCR steps. SMART-Seq v4 libraries were prepared according to manufacturer's instructions accompanying the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech). For library construction of RamDA-seq and C1-RamDA-seq, refer to the protocol in "Single-cell full-length total RNA sequencing uncovers dynamics of non-polyadenylated RNAs, recursive splicing and enhancer RNAs". For rRNA-depleted RNA-seq using 1 and 10 ng of diluted total RNA, sequencing libraries were prepared with KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems) according to the manufacturer's protocol. C1-SMART-Seq v4 libraries were prepared using Fluidigm C1 with the script "Full-length mRNA Sequencing" was downloaded from Script Hub (https://jp.fluidigm.com/c1openapp/scripthub)in accordance with the manufacturer's protocol. For analyses of plate-to-plate variability (batch effect) of RamDA-seq, we prepared RamDA-seq library DNA by using the advanced method of RamDA-seq (refer to the protocol in "Single-cell full-length total RNA sequencing uncovers dynamics of non-polyadenylated RNAs, recursive splicing and enhancer RNAs").
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
FASTQ files were generated using bcl2fastq (v2.16.0.10).
For RamDA-seq and C1-RamDA-seq data with living cells, fastq-mcf (version 1.04.807) was used for adapter trimming with the parameters '-l 36 --lowcomplex-pct 74 --homopolymer- pct 74 -k 4' for RamDA-seq and '-L 75 -l 46 -k 4 -q 30' for C1-RamDA-seq. The reads were mapped to the mouse genome (mm10) using HISAT2 with the parameters '--dta-cufflinks -p 4 -k 5 -X 800 --sp 1000,1000'. Uniquely mapped reads were selected using the bamtools ‘filter’ command with the parameters '-isMapped true -tag NH:1' and samtools ‘view’ command with the parameter ‘-q 40’. BigWig files were generated using deepTools (version 2.2.4). The transcript-level expression levels were quantified using sailfish (version 0.9.2). The transcript-level expression levels were also calculated using Bowtie2 (version 2.2.6) with the parameters '-k 100 -X 800' and eXpress (version 1.5.1) with the parameter '--no-bias-correct'.
For each scRNA-seq method data with 10 pg of diluted RNA, fastq-mcf (version 1.04.807) was used to trim adapter sequences and generate read lengths of 42 nt with the parameters ‘-L 42 -l 42 -k 4 -q 30 -S’. For comparison, we also prepared R1 reads of rdRNA-seq and paRNA-seq data. The reads were mapped to the mouse genome (mm10) using HISAT2 (version 2.0.1) with parameters ‘--dta-cufflinks -p 4 -k 5 --sp 1000,1000’. Uniquely mapped reads were selected using the bamtools (version 2.0.1;) 53 ‘filter’ command with the parameters `-isMapped true -tag NH:1` and the samtools (version 2.0.1)54 ‘view’ command with the parameter ‘-q 40’. BigWig files were generated using deepTools (version 2.2.4). Sailfish (version 0.9.2) was used to quantify transcript-level expressions.
For bulk poly(A) and total RNA-seq data, fastq-mcf was used to trim adapter sequences with the parameters ‘-l 50 --lowcomplex-pct 36 --homopolymer-pct 36 -k 4 -S’. The reads were mapped to the mouse genome (mm10) using HISAT2 with the parameters '--dta-cufflinks --rna-strandness RF -k 5 --no-mixed --no-discordant --sp 1000,1000'. Properly (i.e., convergent read pairs) and uniquely mapped reads were selected using the bamtools ‘filter’ command and samtools ‘view’ command with the parameter ‘-q 40’. For genome-guided transcriptome assembly, Cufflinks (version 2.2.1) was used with the parameters ‘--multi-read-correct --frag-bias-correct -M $mask --library-type fr-unstranded’. A GTF file of tRNA and rRNA annotations in GENCODE (vM9) were provided to mask the genome. Cuffcompare was used to annotate transcripts with transfrag class codes with respect to the GENCODE (vM9) annotation. According to the Cuffcompare class codes, we selected (1) unnannotated transcripts with class codes of "i", "o", "u", "x", or "s" and (2) unannotated splicing variant transcripts (‘j’ class (potentially novel isoform)) with at least one unannotated splice junction. We then removed (1) unannotated transcripts with exons located within 100 bp of the tRNA or rRNA annotations in GECODE or RepeatMasker, or pseudogene annotations in GENCODE (‘2wayconspseudos’), and (2) unannotated transcripts with lengths that were not monger than 200 bp. These filtered unannotated gene models were further merged with gene models in GENCODE vM9.
Genome_build: mm10
Supplementary_files_format_and_content: txt, gtf, bigWig
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| Submission date |
May 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Hiroaki Tateno |
| E-mail(s) |
h-tateno@aist.go.jp
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| Organization name |
National Institute of Advanced Industrial Science and Technology (AIST)
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| Lab |
Biotechnology Research Institute for Drug Discovery (BRD)
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| Street address |
Tsukuba Central 2, 1-1-1 Umezono
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| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
305-8568 |
| Country |
Japan |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE98664 |
Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs |
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| Relations |
| BioSample |
SAMN06916088 |
| SRA |
SRX2789736 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2608096_C1RamDA_PrE_06.bw |
6.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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