|
| Status |
Public on Jan 13, 2017 |
| Title |
mat06_zn06 |
| Sample type |
SRA |
| |
|
| Source name |
Strains ALXC2 x ALXC9
|
| Organism |
Oxytricha trifallax |
| Characteristics |
strain: Strains ALXC2 x ALXC9
developmental stage: 6 hours after mixing mating strains
|
| Treatment protocol |
Mating competent strains ALXC2 and ALXC9 were grown vegetatively to high density, fed lightly the day before a mating and allowed to completely exhaust their food supply. Cells were then cotton filtered to remove any residual algae and were concentrated on 10mM Nitex membranes into Pringhsheim salts media (Fang et al., 2012). Each individual mating strain was counted and cells were then mixed at equal numbers to a total concentration of 1,500 cells per mL in Pyrex dishes. These mating cells were then fed 1 mL of unwashed Klebsiella pneumonia stationary phase culture as a food source. Aggregates of 10-30 cells were observed by 2 hours after mixing mating types, with the first mating pairs visible by 4 hours post mixing. Typical mating efficiency when mixing ALXC2 and ALXC9 strains is ~70%, with visible anlage present by 48 hours.
|
| Growth protocol |
Oxytricha trifallax strains ALXC2 and ALXC9 (Zahler et al., 2012) were grown vegetatively in Pyrex dishes in inorganic salts media (Chang et al., 2004) using the food source Chlorogonium elongatum (UTEX collection strain B203). Typical daily feedings consisted of 10 mL of washed algae per 300 mL Pyrex dish of Oxytricha trifallax, depending on culture density.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Pyrex dishes of mating cells were cotton filtered to remove cellular debri and concentrated onto 10 mM Nitex and transferred into microcentrifuge tubes. Cells were gently pelleted for 2 minutes at 500 x g in a microcentrifuge and supernatants were removed leaving 50 mL of pelleted cells. 200 mL of mirVana Lysis/Binding Buffer from the mirVana miRNA Isolation Kit (Ambion) was added to each tube. Total RNA was purified using the kit’s protocol for total RNA purification. Total RNA yields from a single 300mL Pyrex dish of cells (~450,000 cells) were typically between 100 and 300mg.
mRNA cDNA libraries were prepared using the TruSeq RNA Sample Preparation v2 Kit (Illumina) following the manufacturer’s LT protocol. Library preparation started with 3mg of total RNA from each of the control and mating cell timepoints. Poly-A containing mRNA molecules were selected for using poly-T oligo attached beads with two rounds of purification. Individual libraries were analyzed on a Bioanalyzer (Agilent) for cDNA quality and bar-coded libraries were pooled into one sample for sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Raw sequencing results in the form of FASTQ files were used as input for alignment of the sequencing data to the Oxytricha trifallax macronuclear genome reference RNA database (Swart et al., 2013).
Alignments were performed with Tophat2, which produced BAM alignment files (Kim et al., 2013).
Tophat BAM files were used directly as input into Cuffdiff2 to generate normalized FPKM values (Trapnell et al., 2013).
Genome_build: The reference RNA GTF and macronuclear genome sequence files were retrieved from the Oxytricha genome website (oxy.ciliate.org).
Supplementary_files_format_and_content: tab delimitted table showing the fpkm coverage for each library on each annotated Oxytricha trifallax gene
|
| |
|
| Submission date |
Aug 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Alan M Zahler |
| E-mail(s) |
[email protected]
|
| Phone |
8314595131
|
| Organization name |
UC Santa Cruz
|
| Department |
MCD Biology
|
| Lab |
Zahler Lab
|
| Street address |
Sinsheimer Labs
|
| City |
Santa Cruz |
| State/province |
CA |
| ZIP/Postal code |
95064 |
| Country |
USA |
| |
|
| Platform ID |
GPL15459 |
| Series (1) |
| GSE86081 |
mRNA Expression Profiles of Macronuclear Development in Oxytricha trifallax Implicate Scores of Functionally and Evolutionarily Diverse DNA and RNA Binding Proteins in the Program |
|
| Relations |
| BioSample |
SAMN05687737 |
| SRA |
SRX2054525 |
