|
| Status |
Public on Jan 25, 2018 |
| Title |
RIC_day5_r2 |
| Sample type |
SRA |
| |
|
| Source name |
Ribosome-induced cell clusters (RICs) day 5 replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Ribosome-induced cell cluster (RIC)
|
| Treatment protocol |
Cells were collected using conventional trypsin digestion. The obtained cell suspensions were adjusted to approximately 2.5 × 105 cells/mL in PluriSTEM Human ES/iPS Medium (Merck) and then applied 2 ml to 6-well plate (Thermo Fisher Scientific) containing 20 µl of ribosome solution (1 µg/µl) that is extracted from Escherichia coli JE28. The plated cells were cultured for 14 days, with half the medium replaced every 3 or 4 days.
|
| Growth protocol |
HDFs purchased from Cell Applications were used within 3 passages from culturing of a new vial. HDFs were maintained at 37°C in a humidified atmosphere containing 5% CO2.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
We used the TRIzol reagent (Thermo Fisher Scientific) with 1.0-mm glass beads (WAKENBTECH), a KONTES Homogenizer pestle (Kimble Chase), and a ReliaPrep RNA Cell Miniprep System (Promega) for purification. RNA quality was assessed using a Bioanalyzer (Agilent Technologies).
Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250-300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter). Then 3 μl USER Enzyme (NEB) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
RNA-seq for ribosome-induced cell clusters (RICs) day 5 replicate 2
|
| Data processing |
RTA software version 2.7.7 (Illumina) was used for base calling.
RNA-seq data were filtered using the following criteria: (1) remove reads containing adapters; (2) remove reads containing N > 10%, where N represents the base cannot be determined; (3) remove reads containing low quality (Qscore ≤ 5) base which is over 50% of the total base.
Then, RNA-seq reads were mapped to the human transcriptome using Tophat2 program version 2.1.1 where the genes.gtf file in the Illumina iGenome hg19 was used for the transcriptome annotation.
The number of reads mapped for each gene was counted using HTSeq package version 0.7.2.
For principal component analysis, read counts are transformed into counts per million (CPM) and log2-transformed using edgeR package version 3.16.5.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files that include the number of reads mapped for each gene.
|
| |
|
| Submission date |
May 19, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Yutaka Saito |
| E-mail(s) |
[email protected]
|
| Organization name |
National Institute of Advanced Industrial Science and Technology (AIST)
|
| Street address |
2-4-7 Aomi
|
| City |
Koto-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
135-0064 |
| Country |
Japan |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE99088 |
Ribosome Incorporation into Somatic Cells Promotes Lineage Transdifferentiation towards Multipotency [RNA-Seq] |
| GSE99089 |
Ribosome Incorporation into Somatic Cells Promotes Lineage Transdifferentiation towards Multipotency |
|
| Relations |
| BioSample |
SAMN07142948 |
| SRA |
SRX2835323 |
