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Series GSE98664 Query DataSets for GSE98664
Status Public on Feb 12, 2018
Title Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary We report a novel single-cell total RNA-seq method, RamDA-seq, by combining a novel reverse transcription (RT) technology, RT with random displacement amplification (RT-RamDA), and not-so-random (NSR) primers. RT-RamDA provides global cDNA amplification directly from RNA during RT without any universal adopters, which benefits RT efficiency, simplification of the procedure, avoiding the step of PCR amplification, and decontamination of genomic DNA. NSR enables random priming while preventing cDNA synthesis from rRNAs. RamDA-seq showed high sensitivity to non-poly(A) RNAs and full-length coverage even for extremely long transcripts (>10 kb). Moreover, RamDA-seq revealed recursive splicing, a multi-step, splicing, within > 300 kbp-long introns. Finally, RamDA-seq enabled the first genome-wide analysis of enhancer RNAs (eRNAs) in single cells.
Overall design Total 1,172 single cells (mouse ES, PrE and differentiating cells from ES cells) were sequenced by using RamDA-seq protocol:
(1) Cell cycle samples (277 RamDA-seq samples with mouse ES cells from G1, S, and G2M phases) were sequenced.
(2) Cell differentiation time series RamDA-seq samples (456 RamDA-seq samples with mouse ES cells collected 0, 12, 24, 48, and 72 h after the induction of cell differentiation into PrE cells ) were sequenced.
(3) 55 C1-RamDA-seq samples with mouse ES and PrE cells were sequenced.
(4) To evaluate plate-to-plate variability (batch effect) of RamDA-seq, two experimenters performed RamDA-seq with two plates (384 cells) of single-cell samples (consisting of mouse ES and PrE cells) on two separate days.

For assesment of RamDA-seq, 9 averaged single-cell lysate samples and 16 purified total RNA samples diluted to 10 pg were sequenced by using RamDA-seq protocol. We also sequenced 4 SMART-Seq v4 samples using 10 pg of total RNA. Moreover, 6 poly(A) RNA-selected and 6 rRNA-depleted samples derived from 1 ug of total RNA were sequenced by using a commercially available library prepararion kit. Furthermore, to evaluate reproducibility, we sequenced ~96 diluted RNA samples (corresponding to the entire plate) with RamDA-seq, C1-RamDA-seq, and C1-SMART-seq v4.

To compare RamDA-seq and rRNA-depleted RNA-seq prepared with equal amounts of input RNA, we sequenced RamDA-seq samples using 1 ng of diluted total RNA and rRNA-depleted RNA-seq samples using using 1 and 10 ng of diluted total RNA.
Contributor(s) Hayashi T, Ozaki H, Sasagawa Y, Nikaido I
Citation(s) 29434199
Submission date May 08, 2017
Last update date Jul 25, 2021
Contact name Hiroaki Tateno
Organization name National Institute of Advanced Industrial Science and Technology (AIST)
Lab Biotechnology Research Institute for Drug Discovery (BRD)
Street address Tsukuba Central 2, 1-1-1 Umezono
City Tsukuba
State/province Ibaraki
ZIP/Postal code 305-8568
Country Japan
Platforms (2)
GPL17021 Illumina HiSeq 2500 (Mus musculus)
GPL19057 Illumina NextSeq 500 (Mus musculus)
Samples (1507)
GSM2608052 NEB_ES0_01
GSM2608053 NEB_ES0_02
GSM2608054 NEB_ES0_03
BioProject PRJNA385832
SRA SRP106644

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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE98664_RAW.tar 14.5 Gb (http)(custom) TAR (of BW)
GSE98664_count_sailfish_GENCODEvM9_10pg_10M.csv.gz 6.6 Mb (ftp)(http) CSV
GSE98664_count_sailfish_GENCODEvM9_ERCC_RamDA_Large_volume.txt.gz 4.5 Mb (ftp)(http) TXT
GSE98664_count_sailfish_GENCODEvM9_ERCC_mESC_10pg_RamDA_C1RamDA_C1SMARTseqV4.txt.gz 33.4 Mb (ftp)(http) TXT
GSE98664_mergedGTF.gtf.gz 26.9 Mb (ftp)(http) GTF
GSE98664_tpm_sailfish_GENCODEvM9_C1RamDA_cells.txt.gz 8.0 Mb (ftp)(http) TXT
GSE98664_tpm_sailfish_GENCODEvM9_ERCC_RamDA_Large_volume.txt.gz 4.7 Mb (ftp)(http) TXT
GSE98664_tpm_sailfish_GENCODEvM9_RamDA_Batch_effect_mES_PrE.txt.gz 46.2 Mb (ftp)(http) TXT
GSE98664_tpm_sailfish_GENCODEvM9_RamDA_cells.txt.gz 123.5 Mb (ftp)(http) TXT
GSE98664_tpm_sailfish_mergedGTF_10pg_7.5M.txt.gz 5.4 Mb (ftp)(http) TXT
GSE98664_tpm_sailfish_mergedGTF_RamDA_mESC_differentiation_time_course.txt.gz 99.6 Mb (ftp)(http) TXT
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Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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