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| Status |
Public on Aug 26, 2020 |
| Title |
YH4A |
| Sample type |
SRA |
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| Source name |
yeast cells
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| Organism |
Cryptococcus neoformans var. grubii H99 |
| Characteristics |
tissue: yeast cells
medium: YPD
temperature: 37C
growth time mins.: 120
biological replicate: A
tagid: Tag02
tagseq: AAGTGCCGT
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| Growth protocol |
- Grow a 10mL culture of C. neoformans in YPD for 5 days.
- In duplicate, pre-warm 2x 100mL each of YPD at 25C, YPD at 37C, RPMI + serum at 25C, RPMI + serum at 37C, in incubators shaking at 60rpm.
- Fix and freeze 3mL of stationary-phase culture:
- Place 4 x 3mL of one stationary-phase culture in separate falcon tubes.
- Resuspend each cell pellet in a different pre-warmed medium condition (YPD at 25C, YPD at 37C, RPMI + serum at 25C, RPMI + serum at 37C), and start stopwatch. Transfer to 100mL total in shaking flasks in appropriate incubator as soon as possible.
- At 10, 30, 60 and 120 minutes after inoculation, fix and freeze 10mL from each condition, as above.
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| Extracted molecule |
total RNA |
| Extraction protocol |
- Fix and freeze yeast culture:
- Pre-chill a 50mL tube with 6mL 100% Methanol on dry ice (tightly capped so CO2 does not dissolve in MetOH).
- Add 10mL of culture to the chilled MetOH, cap tube, shake to mix, place on dry ice for up to 5 min.
- Spin 2 min at ~2500g, pour off supernatant.
- Resuspend cell pellet in 1mL ice-cold H2O and transfer to 2mL screw-cap tube.
- Spin 1min at 5000g, remove supernatant carefully, and freeze pellet in sealed tube at -80C.
- Freeze-dry the pellet in a lyophilizer until dry.
- Extract RNA using the RNAeasy plant and fungi kit (Qiagen)
RNAtagseq essentially as described in Shishkin et al., Nat. Methods 2015, "Simultaneous generation of many RNA-seq libraries in a single reaction", doi: 10.1038/nmeth.3313. Edits to protocol at https://github.com/ewallace/protocols/blob/master/RNAtagseq.md
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Base-calling using illumina basespace
Split files by RNA-tag using initial 8nt Tagseq barcode using SplitRNATagSeqFastQ.py https://github.com/ewallace/pyRNATagSeq.
Concatenate files with same tag/index combination from distinct lanes.
Further processing with CutDedupTrimQuant.py from 5' adapter trimming with flexbar, then deduplication of 3' 5nt UMIs with DedupEndFastQ.py, then trimming of the UMI and quality filtering with flexbar
kallisto to quantify reads (counts/TPMs) per gene
Genome_build: CNA3
Supplementary_files_format_and_content: Collated TPMs across all samples and genes (single transcript, no isoforms.
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| Submission date |
Jun 20, 2019 |
| Last update date |
Aug 26, 2020 |
| Contact name |
Edward William Joseph Wallace |
| E-mail(s) |
edward.wallace@ed.ac.uk
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| Organization name |
University of Edinburgh
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| Department |
Institute for Cell Biology, School of Biological Sciences
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| Street address |
Waddington Building, Max Born Crescent
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3BF |
| Country |
United Kingdom |
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| Platform ID |
GPL24689 |
| Series (1) |
| GSE133067 |
Cryptococcus neoformans reactivation in different media and temperatures |
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| Relations |
| BioSample |
SAMN12098578 |
| SRA |
SRX6098075 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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