|
| Status |
Public on Nov 28, 2019 |
| Title |
Mkt1 CRAC 2 |
| Sample type |
SRA |
| |
|
| Source name |
Mkt1 CRAC
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: h+ mkt1-HTP-KanR otr1R(Sph1):ade6+ ade6-210 leu1-32 ura4D18
|
| Treatment protocol |
Cells were UV-irradiated in the X-Varilinker (Van Nues et al Nature Communications 2017, doi: 10.1038/s41467-017-00025-5) for 42.5 seconds in PMG minus Trp medium and harvested by filtration. Cells were snap-frozen in liquid nitrogen and stored at -80ºC.
|
| Growth protocol |
Cells were grown to exponential phase in PMG minus TRP medium at 32ºC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells (0.8-1g) were lysed in 1V (w/v) of TN150 (50 mM Tris pH 8.0, 0.1% NP-40, 150 mM NaCl, 5 mM beta-mercaptoethanol, Roche protease inhibitor cocktail) with 3V of 0.5 mm Zirkonia beads by vortexing for five minutes at full speed. Lysates were clarified by centrifugation and processed as previously described (Granneman et al PNAS 2009, doi: 10.1073/pnas.0901997106).
Libraries were constructed using the CRAC library preparation method described in Granneman et al PNAS 2009 with small modifications to the adapter sequences (see sequences below)
PCR oligonucleotides for making CRAC libraries
3' adapter:
App_PE: 5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’
5' adapters:
L5Ba: 5'-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrArGrArGrC-OH-3' (Mkt1 CRAC 1)
L5Bd: 5'-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrUrCrUrCrUrArGrCrN-OH-3' (no-tag negative control CRAC 1)
L5Aa: 5'-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrUrArArGrC-OH-3' (Mkt1 CRAC 2)
L5Ac: 5'-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrGrCrGrCrArGrC-OH-3' (no-tag negative control CRAC 2)
P5_forward: 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
P3_reverse: 5’-CAAGCAGAAGACGGCATACGAGAT(6nt barcode)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
cross-linked RNA
161108_K00166_0147_BHFT27BBXX_8_IL-PE-002_*_NNNAGAGC_L5Ba-M4
|
| Data processing |
Processing steps were performed using the pyCRAC package version 1.2.2.7; Demultiplexing was performed using pyBarcodeFilter.py.
3' adapter removal and 3' end quality trimming (min Phred = 30) was performed using FlexBar (version 2.7).
Reads were collapsed using random nucleotides present in 5' barcodes using pyFastqDuplicateRemover.py.
tRNA-reads in fasta files were mapped by STAR (version 2.7.1a) and unmapped output used for further analysis steps which were done with pyCRAC version 1.4.5
Reads were mapped to the S. pombe reference genome (v2.30; source pombase) using novoalign (version 3.09.02) and feature counts were generated using pyReadCounters (version 1.4.4.) with options –blocks --mutations=delsonly
The gtf files were used to generate bedgraph files for visualisation.
Genome_build: ASM294v2.30
Supplementary_files_format_and_content: gtf and bedgraph files containing genomic positions of mapped reads after tRNA reads have been filtered out
|
| |
|
| Submission date |
Aug 12, 2019 |
| Last update date |
Nov 29, 2019 |
| Contact name |
Elizabeth H Bayne |
| Organization name |
University of Edinburgh
|
| Department |
School of Biological Sciences
|
| Street address |
Alexander Crum Brown Road
|
| City |
Edinburgh |
| ZIP/Postal code |
EH9 3FF |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL22682 |
| Series (1) |
| GSE135735 |
Mkt1 is required for RNAi-mediated silencing and establishment of heterochromatin in fission yeast [CRAC] |
|
| Relations |
| BioSample |
SAMN12562126 |
| SRA |
SRX6706724 |
