|
| Status |
Public on Dec 01, 2020 |
| Title |
anti-Myc IP of Myc-EhAgo2-2, nuclear lysate [IPAgo2_nuc_RppH] |
| Sample type |
SRA |
| |
|
| Source name |
E. histolytica trophozoites
|
| Organism |
Entamoeba histolytica |
| Characteristics |
strain/genotype: HM-1:IMSS,Myc-EhAgo2-2
development stages: trophozoites
treatment: IP-ed RNA, cloned use RppH
isolation method: IP-ed RNA isolated by Trizol
molecule subtype: small RNA
|
| Treatment protocol |
The cell lysates for transfected cells: parasites were harvested by chilling on ice, spun down, and washed 1x in cold PBS and lysed by lysis buffer contains 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 10% (v/v) glycerol and 50 mM NaCl, IGEPAL CA-630 (equivalent of NP-40) at 0.5% (v/v) plus 1mM NaF, 1mM DTT, 1mM PMSF and HALT EDTA free protease inhibitors (100x stock diluted to 2x)(Thermo Scientific) and RNase inhibitor (1 unit/ml).
|
| Growth protocol |
E. histolytica trohozoites were grown in TYI at 37°C; E. invadens was grown in standard axenic culture conditions (LYI @ 25°C); E. histolytica overexpressing cell lines are kept at 6μg/ml G418.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Parasites were harvested by chilling on ice, spun down, and washed 1x in cold PBS. RNA was isolated using Trizol (Invitrogen) following the manufacturer's protocol.
We used the NEBNext multiplex small RNA library prep set for Illumina (NEB#E7300S) and followed the manufacturer’s protocol with the following modifications: total RNA (25-50 µg) was first fractioned for the size 15-30nt RNA or 30-45nt RNA using 12% polyacrylamide urea gel. The size-selected RNA was then treated with tobacco acid pyrophosphatase [TAP] to convert 5’ poly-P to 5’ single-P (Epicentre) or RppH (NEB). The RNA sample was then fed into the pipeline as described in the manufacturer's protocol. Once the cDNA was made, the barcode primers were incorporated into each sample using PCR with kit’s provided barcoded primer; typically we used 8-12 PCR cycles to get a clear final PCR product band. The final PCR product was quantified using Nanodrop, and samples were pooled and shipped to the collaborator for Illumina sequencing. The platform MiSeq (Illumina) was used, the number
Sequencing of E. histolytica small RNAs for size-selected (30-45nt) total RNA of E. histolytica trophozoites; from size-selected (30-45nt) total RNAs of development of E. invadens; from IP-ed RNAs of EhAgo proteins and mutant.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Basecalling was performed using Illumina Sequencing Data Analysis software
Adaptor sequences were removed from each read
Duplicate sequences were removed
Unique reads were filtered by removing all reads that mapped to genomic tRNA, rDNA, LINES and SINES
Filtered reads were mapped to the genome using bowtie with the parameters: -v 1 --all
Genome_build: E. histoyiitca HM-1:IMSS version 1.3 (http://amoebadb.org);E. invadens IP-1 version 1.3 (http://amoebadb.org)
Supplementary_files_format_and_content: txt files are bowtie output
|
| |
|
| Submission date |
Sep 09, 2020 |
| Last update date |
Dec 01, 2020 |
| Contact name |
Gretchen Marie Ehrenkaufer |
| E-mail(s) |
[email protected]
|
| Organization name |
Stanford Uninversity
|
| Department |
Microbiology and Immunology
|
| Lab |
Singh
|
| Street address |
Grant S-143, 300 Pasteur Dr.
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL19933 |
| Series (1) |
| GSE157756 |
Non-templated oligo-adenylation of small RNAs in the parasite Entamoeba |
|
| Relations |
| BioSample |
SAMN16091615 |
| SRA |
SRX9102554 |
