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| Status |
Public on Oct 27, 2018 |
| Title |
TPT |
| Sample type |
SRA |
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| Source name |
laryngeal squamous carcinoma cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Laryngeal squamous carcinoma cell line TU-177
cell surface antigen: unsorted TU-177 cells
isolation method: none
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| Treatment protocol |
Cells positive or negative for CD44 and CD133 surface markers were isolated from cultured TU-177 cells by MACS by using CD44 MicroBeads or CD133-PE antibody (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer’s instructions
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| Growth protocol |
TU-177 cells were cultured in MEM supplemented with 10% fetal bovine serum.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by using Trizol reagent (Invitrogen) according to the manufacturer’s instructions.
A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Firstly, ribosomal RNA was removed by Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol precipitation. Subsequently, sequencing libraries were generated using the rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Briefly, 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(RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. 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 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) 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, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Raw data(raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data(clean reads) were obtained by removing reads containing adapter, reads on containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the down stream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using bowtie2 v2.2.8 and paired-end clean reads were aligned to the reference genome using HISAT2(Langmead, B.et al) v2.0.4. HISAT2 was run with ‘--rna-strandness RF’, other parameters were set as default.
The mapped reads of each sample were assembled by StringTie (v1.3.1) (Mihaela Pertea.et al. 2016) in a reference-based approach. StringTie uses a novel network flow algorithm as well as an optional de novo assembly step to assemble and quantitate full-length transcripts representing multiple splice variants for each gene locus.
Transcripts predicted with coding potential by either/all of the four tools (CNCI, CPC, Pfam-sca, phyloCSF) were filtered out, and those without coding potential were our candidate set of lncRNAs.
Cuffdiff (v2.1.1) was used to calculate FPKMs of both lncRNAs and coding genes in each sample (Trapnell, C. et al. 2010). Gene FPKMs were computed by summing the FPKMs of transcripts in each gene group. FPKM means fragments per kilo-base of exon per million fragments mapped, calculated based on the length of the fragments and reads count mapped to this fragment.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM/TPM values for each Sample
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| Submission date |
Oct 27, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Yongyan Wu |
| E-mail(s) |
wu-yongyan@163.com
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| Organization name |
Shanxi Medical University
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| Department |
Department of Otolaryngology Head & Neck Surgery, The First Hospital
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| Lab |
Shanxi Key Laboratory of Otorhinolaryngology Head and Neck Cancer
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| Street address |
No.85 South Jiefang Road, Taiyuan, Shanxi, China
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| City |
Taiyuan |
| State/province |
Shanxi |
| ZIP/Postal code |
030001 |
| Country |
China |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE106283 |
Whole transcriptomic sequencing of CD133+CD44+ cancer stem cells from laryngeal squamous carcinoma cell line |
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| Relations |
| BioSample |
SAMN07842269 |
| SRA |
SRX3338992 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2834569_TPT_circRNA_TPM.txt.gz |
16.0 Mb |
(ftp)(http) |
TXT |
| GSM2834569_TPT_lncRNA_FPKM.txt.gz |
93.7 Kb |
(ftp)(http) |
TXT |
| GSM2834569_TPT_mRNA_FPKM.txt.gz |
1.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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