|
| Status |
Public on Dec 31, 2023 |
| Title |
lkh1 mutant basal sample_2 |
| Sample type |
SRA |
| |
|
| Source name |
lkh1 mutant basal sample
|
| Organism |
Cryptococcus neoformans var. grubii H99 |
| Characteristics |
genotype: lkh1<delta> mutant
treatment: none
time point: basal
|
| Treatment protocol |
50 ml of cells were harvested for the basal sample and rapamycin (3 ng/ml) was treated to the remaining cells. After rapamycin treatment, cells were further incubated at 30℃ for 60 mins.
|
| Growth protocol |
Strains were grown in a 30 ml liquid YPD medium for 16 hr at 30℃. Then, overnight culture was inoculated into 100 ml fresh YPD medium and adjusted to OD600=0.2. The cells were further incubated until OD600 reached approximately 0.6.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
1. Freeze cells in Liquid Nitrogen for 30min. 2. Lyophilize samples overnight. 3. Add 3ml of 3mm glass beads and vortex vigorously until fine powder is created. 4. Add 4ml of Easyblue (dissolve powder completely) and let them sit 5 minutes. 5. Add 800 ul of chloroform (invert 10 times). 6. Let them sit 3 minutes and transfer to 14 ml round bottle tubes. 7. Spin at 10,000 rpm for 15 minutes. 8. Transfer supernatant to new round-bottom tubes. 9. Add 1.6 ml of isopropanol and capping and gently invert. 10. Store for 10 minutes at room temperature. 11. Spin for 10 minutes at 10,000 rpm. 12. Pour off supernatant. 13. Add 4 ml of 75% RNase free-EtOH (made with DEPC dH2O) and invert 4-5 times. 14. Spin for 5 minutes at 8,000 rpm. 15. Pour off EtOH (Let tubes air-dry upside down). 16. Resuspend in appropriate volume of DEPC dH2O. 17. Heat at 55℃ for 10 minutes. 18. Transfer RNA to microtube and measuring the concentration. 19. For purification of the extracted total RNA, we used RNeasy spin column (Qiagen) following the manufacturer’s protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Illumina bcl2fastq2-v2.20.0 software used for basecalling.
Trimmomatic program is used to remove adapter sequences and bases with base quality lower than three from the ends. Also using sliding window method, bases of reads that does not qualify for window size 4, and mean quality 15 are trimmed. Afterwards, reads with length shorter than 36bp are dropped to produce trimmed data.
Sequenced reads were mapped to "GCF_000149245.1_CNA3" whole genome by using Bowtie version 1.1.2 with parameter -S -m 1 -I 0 -X 500 --fr --max --un -a --best -1 {read1} -2 {read2}
FPKM were calculated by using HTseq (htseq-count) version 0.10.0, with parameter -f bam -r name -t exon -i gene_id -m intersection-nonempty -s reverse -o {output} --nonunique none {alignment_file(.bam)} {gff/gtf_file}
Assembly: GCF_000149245.1_CNA3
Supplementary files format and content: *_FPKM.txt: Tab-delimited text files include FPKM values for each Sample.
|
| |
|
| Submission date |
May 26, 2023 |
| Last update date |
Dec 31, 2023 |
| Contact name |
Sunhak Kwon |
| E-mail(s) |
shkwon10@kaeri.re.kr
|
| Phone |
01031359650
|
| Organization name |
Korea Atomic Energy Research Institute
|
| Street address |
29, Geumgu-gil
|
| City |
Jeongeup-si |
| ZIP/Postal code |
56212 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL33437 |
| Series (1) |
| GSE233612 |
Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans |
|
| Relations |
| BioSample |
SAMN35441515 |
| SRA |
SRX20526889 |
