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| Status |
Public on Jun 28, 2024 |
| Title |
RNA bound with hPAC |
| Sample type |
SRA |
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| Source name |
WT
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293F
cell type: human embryonal kidney (HEK) cells
genotype: WT
treatment: transient expression of wild type human acid-sensitive outwardly rectifying channel ASOR
time: Day 3
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| Treatment protocol |
(1) For expression xPAC, recombinnant baculovirus were generated following the standard protocol of Bac-to-Bac Expression Systems (Invitrogen), Sf9 cell (cell density: 1.5-2.0 ×10^6 cells/ml) were infected with P2 virus, and incubated at 27 °C for 60 hours. (2) For expression hPAC, expression plasmids were transfected into HEK293F cells (cell density: 1.5-2.0 ×10^6 cells/ml) using PEI (25 kDa, Polysciences Inc.). The transfected cells were incubated at 37 °C for 60 hours.
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| Growth protocol |
(1) Sf9 cells were maintain in SIM SF Expression Medium (Sino Biological Inc.) with 120 rpm at 27°C in a shaker. (2) HEK293F cells were maintained in SMM 293T-II medium (Sino Biological Inc.) at 37 °C, 5% CO2, and 130 rpm shaking.
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| Extracted molecule |
other |
| Extraction protocol |
(1) The xPAC was expressed in Sf9 and the cells were centrifuged at 800 × g for 15 min and suspended in lysis buffer (25 mM Tris, 150 mM NaCl, pH 8.0) with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.8 µM aprotinin, 2 µM pepstatin A and 5 µg/mL leupeptin). The collected cells were flash-frozen in liquid nitrogen and stored at −80 °C before purification. The thawed cells were solubilized by adding 2% (w/v) n-Dodecyl β-D-maltoside (DDM, Anatrace) and stirring at 4 °C for 2 h. The resulting cell lysate was centrifuged at 27,000 × g at 4 °C for 30 min. The supernatant was incubated with Strep-tactin resin (GE Healthcare) at 4 °C for 1 h. The resin was washed with lysis buffer supplemented with either 0.06% (w/v) digitonin (Biosynth). Proteins were eluted with elution buffer (25 mM Tris, 150 mM NaCl, 5 mM desthiobiotin, pH 8.0) containing with 0.06 % digitonin. The protein samples were further purified by size-exclusion chromatography (SEC, Superose 6 increase 10/300 GL, GE Healthcare) in lysis buffer containing 0.06% digitonin. The purified xPAC proteins were used to extract RNA by using a phenol-chloroform extraction followed by ethanol precipitation. (2) The hPAC was expressed in HEK293F cells. The purification procedure was similar to that described for xPAC, with the following modification. The wash buffer contained 25 mM Tris pH 8.0, 150 mM NaCl, and 0.05% DDM. The protein was eluted with elution buffer containing 25 mM Tris, pH 8.0, 150 mM NaCl, 0.05% DDM, and 5 mM desthiobiotin. The RNAs enriched in the hPAC samples were extracted using the phenol-chloroform method followed by ethanol precipitation.
The purified RNA was demethylated by rtStarTM tRF&tiRNA Pretreatment Kit (Arraystar, AS-FS-005) and partially hydrolyzed according to Hydro-tRNAseq mothod. The library was prepared by using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® kit (New England Biolabs, E7300L/E7850L)
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The quality of sequence data was checked by FastQC software (v0.11.8) .
(1) For data from xPAC sample, 5’adaptor and 3’-adaptor trimmed reads were aligned to the reference genome of Spodoptera frugiperda (fall armyworm) using STAR (v2.7.1a) with default parameters and the NCBI annotation version 1.0 was used (https://www.ncbi.nlm.nih.gov/assembly/GCF_011064685.1/). The 1.24 million reads including unique and multiple mapped reads were retained. The gene expression was quantified using the HTSeq (v0.11.2) with parameters of “-s yes” and “--nonunique all”. Genes with raw counts of less than 10 reads were filtered out. Gene biotypes and tRNA types were from the gene transfer format (GTF) file.
(2) For data from the hPAC sample, 5’-adaptor and 3’-adaptor trimmed reads were aligned to the reference genome of hg38 using BWA (0.7.17-r1198-dirt) with default parameters, and the NCBI annotation version 14 was used (https://www.ncbi.nlm.nih.gov/genome/?term=GRCh38.p14). A total of 1.7 million reads including unique and multiple mapped reads were retained. The gene expression was quantified using the HTSeq (v0.11.2) with parameters of “-s yes” and “--nonunique all”. Gene biotypes and tRNA types were from the GTF file.
The gene expression was quantified using the HTSeq (v0.11.2) with parameters of “-s yes” and “--nonunique all”.
Genes with raw counts of less than 10 reads were filtered out.
Assembly: (1) Spodoptera frugiperda version 1.0; (2) Homo sapiens reference genome GRCh38.p14.
Supplementary files format and content: The tab-delimited text files include gene biotypes, gene raw counts and RPKM values for each sample
Library strategy: Hydro-tRNAseq
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| Submission date |
Jun 13, 2024 |
| Last update date |
Jun 28, 2024 |
| Contact name |
pengliang chi |
| E-mail(s) |
chipengliang@stu.scu.edu.cn
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| Phone |
18428359657
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| Organization name |
Sichuan University
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| Street address |
No.17 People's South Road
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| City |
Chengdu |
| State/province |
Sichuan province |
| ZIP/Postal code |
610041 |
| Country |
China |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE216637 |
Molecular Insights into the Inhibition of Proton-Activated Chloride Channel by Transfer RNA |
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| Relations |
| BioSample |
SAMN41826365 |
| SRA |
SRX24921064 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8328750_hPAC_bound_RNA.txt.gz |
25.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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