|
| Status |
Public on Mar 13, 2021 |
| Title |
KN99 - sap18 day 1, replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
KN99 cells
|
| Organism |
Cryptococcus neoformans |
| Characteristics |
strain: KN99
genotype: cnag_04679{delta}:: NEO
|
| Growth protocol |
Two-liter liquid cultures of all strains were grown in YPAD medium (Difco) by inoculation at low density (0.002-0.004 OD 600 nm) followed by overnight growth with shaking 30C. For RNA-seq experiments, cells were harvested at OD600 of ~ 1
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were lyophilized and then treated with Trizol and agitated with silica beads in an Omni bead mill homogenizer. RNA was extracted from whole cell extract by phase separation with 1 volume chloroform to produce a phenol:chloroform ratio of 1:1, and then washed with 1 volume chloroform. RNA was precipitated in 50% isopropanol, then recovered by centrifugation for 30 min at 18400xg at 4 C and washed with 70% ethanol. RNA from whole cell extract was polyA selected with Streptavidin MagneSphere® Paramagnetic Particles (Promega) and DNase treated with RNA Clean & Concentrator Kit 5 (Zymo).
RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina and mixed before being run over multiple lanes
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
remove adapter with cutadapt 2.4 with Python 3.5.2 cutadapt -a AGATCGGAAGA -A AGATCGGAAGA --trim-n -m 25 -q 10
concatenate files for first direction and second direction from multiple lanes run on same day.
Run STAR_2.5.3 genome generate: STAR --runThreadN 15 --runMode genomeGenerate --genomeDir /data/jade/Data/RNA-seq_Data/STAR/STARindex --genomeFastaFiles /data/jade/H99/150908_H99chr.fasta --sjdbGTFfile /home/jordan/GENOMES/CNA3_all_transcripts.gff3 --sjdbGTFtagExonParentTranscript Parent --sjdbOverhang 99
Align read to genome with 2-pass STAR: STAR first pass with --alignIntronMin 20 --alignIntronMax 2000 --outSJfilterReads Unique, run STAR second pass with --alignIntronMin 20 --alignIntronMax 2000 --outSJfilterReads Unique --outSAMstrandField intronMotif --outFilterMultimapNmax 1 -sjdbFileChrStartEnd [all .tab files from first pass]
count transcripts with DEseq2: htseq-count -f bam -s reverse -t gene -i ID
Identify splice isoforms with JUM_2.0.2: Step A: --JuncThreshold 5 --Condition1_fileNum_threshold 2 --Condition2_fileNum_threshold 2 --IRthreshold 5 --Readlength 100 --Thread 20 Step B: --Test pvalue --Cutoff 1 --TotalFileNum 4 Step C: --Test pvalue --Cutoff 1 --TotalCondition1FileNum 2 --TotalCondition2FileNum 2 --REF CN_refGene_correct.txt
Supplementary_files_format_and_content: txt files from JUM contain information as described https://github.com/qqwang-berkeley/JUM/wiki/2.2-Output-files-(v2.0-and-up)
|
| |
|
| Submission date |
Mar 12, 2021 |
| Last update date |
Mar 13, 2021 |
| Contact name |
Hiten Madhani |
| Organization name |
UCSF
|
| Department |
Biochemistry
|
| Lab |
Madhani Lab
|
| Street address |
600 16th St
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL21073 |
| Series (1) |
| GSE168814 |
HMJSL C. Neoformans splicing factor knockout RNA-seq |
|
| Relations |
| BioSample |
SAMN18287574 |
| SRA |
SRX10332259 |
