|
| Status |
Public on Mar 03, 2019 |
| Title |
307-1 emptyNor1 |
| Sample type |
SRA |
| |
|
| Source name |
Mucosa
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Normal oral keratinocytes
genotype: empty vector pWPI
|
| Treatment protocol |
293T cells were co-transfected by the respective pWPI lentiviral expression plasmids, packaging plasmid psPAX2 (Addgene #12260) and enveloping plasmid pMD2.G (Addgene #12259; gift from Didier Trono) using 25kD linear polyethylenimine (Polysciences). 48h after transfection, supernatant was collected, filtered and applied to NOK cells in the presence of 10 µg/ml polybrene (Santa Cruz). Medium was refreshed after 24h and cells were kept in medium supplemented with 1µg/ml puromycin (Sigma-Aldrich) for selection until all untransduced control cells were dead. Only cells within 10 passages after selection were used for experiments.
|
| Growth protocol |
NOKs pWPI cells and NOKs HPV16E6E7 were kept in Keratinocyte-SFM medium (Life Technologies) containing rhEGF and bovine pituitary gland extract (Life Technologies) for daily cultivating and passaging. Cells were switched into FAD medium containing 75% Ham´s F-12, 25% Dulbecco modified Eagle’s medium (DMEM), 5% fetal bovine serum (FBS), insulin (5 μg/ml), epidermal growth factor (10 ng/ml), cholera toxin (8.4 ng/ml), adenine (24 μg/ml) and hydrocortisone (0.4 μg/ml) for one passage before using for experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNase-Free DNase Set (Qiagen) was used to remove residual DNA for downstream applications. RNA concentrations were determined by NanoDrop™ and 1 µg of RNA was reverse transcribed by RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific) and oligo dT22 primers (Thermo Fisher Scientific) according to the manufacturer's instructions.
Illumina TruSeq Stranded RNA was used according to Illuminas protocols and sequenced on Hiseq 2000 v4
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina bcl2fastq 2.19.0.316 was used for base calling
bowtie2 (v2.2.4) and HISAT2 (v2-2.1.0) were used for alignment, Salmon (v0.8.2) was used for pseudo-alignment
StringTie (v1.3.3b) was used for transcrip assembly
DESeq2 (v1.16.1) and sleuth (v0.29.0) were used for differential expression analysis
Supplementary_files_format_and_content: Processed data files were Excel files including log2fc and q-value of genes
Supplementary_files_format_and_content: BtDe full.xlsx: generated by bowtie2+DESeq2
Supplementary_files_format_and_content: HsStDe_gene_1.xlsx: generated by HISAT2+StringTie+DESeq2
Supplementary_files_format_and_content: SmDe_1.xlsx: generated by Salmon+DESeq2
Supplementary_files_format_and_content: SmSu_wt_1.xlsx: generated by Salmon+sleuth
Supplementary_files_format_and_content: *counting.txt: BtDe counts file
Supplementary_files_format_and_content: *data_stringtie_count.ctab.txt: HsStDe counts file
Supplementary_files_format_and_content: *quant_salmon.sf.txt: SmDe & SmSu counts file
|
| |
|
| Submission date |
Dec 26, 2018 |
| Last update date |
Mar 03, 2019 |
| Contact name |
Martina Niebler |
| E-mail(s) |
m.niebler@dkfz.de
|
| Organization name |
German Cancer Research Center
|
| Street address |
Im Neuenheimer Feld 242
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE124357 |
Transcriptome of human keratinocytes with or without HPV16 oncogene expression |
|
| Relations |
| BioSample |
SAMN10644147 |
| SRA |
SRX5180014 |
