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| Status |
Public on Jul 25, 2023 |
| Title |
Tpd pollen total RNA fertile rep3 |
| Sample type |
SRA |
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| Source name |
pollen
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| Organism |
Zea mays subsp. mays x Zea mays subsp. mexicana |
| Characteristics |
tissue: pollen
genotype: tpd1;tpd2
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| Growth protocol |
The TPD lineage traces to teosinte mexicana collected near Copándaro, Michoacán, Mexico in December 1993. Gamete a, plant 4 of collection 107 was used in an initial outcross to the Mid-western US dent inbred W22 and subsequently backcrossed. Tpd1;Tpd2 (BC8S3) homozygous lines were used for whole-genome sequencing and de novo genome assembly. All additional experiments were performed using Tpd1/tpd1; Tpd2/tpd2 (BC11-BC13) plants or populations derived from maternal segregation of these lines. The lbl1-rgd1 and dcl2-mu1 alleles were backcrossed to W22 ≥ 4 times. All genetic experiments used segregating wild-type progeny as experimental controls. Plants were grown under greenhouse and field conditions. In the greenhouse, starter flats were transplanted to pots containing divot mix and a single scoop of Osmocote 19-6-12. Once established, pots were on an irrigation drip of 20-20-20 at 0.8 EC (200 PPM Nitrogen) with a weekend water flush. Marathon was added as a drench and biweekly sprays of insecticides were applied as needed. Long day (6:00 AM until 10:00 PM) conditions were maintained at a 28˚C set point with cooling to 24˚C at night.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was collected, snap frozen in liquid nitrogen, and stored at -80˚C. Samples were ground into a fine powder using a mortar and pestle on liquid nitrogen. 800ul of pre-extraction buffer (100mM Tris-HCl pH 8.0, 150mM LiCl, 50mM EDTA pH 8.0, 1.5% v/v SDS, 1.5% 2-mercaptoethanol) was added and mixed by vortexing. 500ul of acid phenol:chloroform (pH 4.7-5.0) was added and samples were mixed then spun down at 13,000 x g for 15 minutes at 4˚C. The aqueous layer was extracted and 1ml Trizol per 200mg input tissue of was added. Samples were mixed by vortex and incubated at RT for 10 minutes. 200ul chloroform per 1ml Trizol was added and samples were mixed by vortexing then incubated at RT for 2 minutes. Samples were then spun down at 13,000 x g for 15 minutes at 4˚C. The aqueous phase was extracted and cleaned up using the Zymo RNA Clean and Concentrator-5 kit (Zymo Research, cat#R1013). Only samples with RNA integrity (RIN) scores ≥9 were used for qPCR and sequencing.
Five biological replicates were prepared for each biological condition (Tpd1/tpd1; Tpd2/tpd2 and tpd1; tpd2 siblings). 5ug of total RNA was ribosome depleted using the RiboMinus Plant Kit (ThermoFisher, cat#A1083808), and libraries were prepared using the NEXTFLEX Rapid Directional RNA-seq kit (PerkinElmer, cat#NOVA-5138-08). The size distribution of completed libraries was assessed using an Agilent Bioanalyzer, and quantification was performed using a KAPA Library Quantification kit (Roche, cat#KK4824). Libraries were sequenced on a NextSeq500 platform using a 2x150bp high output run.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
total-rna.fertile.bdg.gz
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| Data processing |
Trimmed reads were aligned to the W22 reference with STAR in two-pass alignment mode. Read counts were assigned to annotated features using featureCounts. For transposable element expression, multi-mapping reads were assigned fractional counts based on the number of identical alignments. Differential expression analysis was performed using edgeR. To avoid false positives, a stringent cutoff (log2FC ≥ 2, FDR ≤ 0.001) was used to call differentially expressed genes. Gene ontology (GO) analysis (Fisher’s exact test, p<0.01) was performed using topGO, and the results were visualized using rrvgo. For data visualization, alignment files were converted to a strand-specific bigwig format using deepTools.
Assembly: Zm-W22-REFERENCE-NRGENE-2.0
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| Submission date |
Jun 14, 2023 |
| Last update date |
Jul 25, 2023 |
| Contact name |
Robert A Martienssen |
| E-mail(s) |
martiens@cshl.edu
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| Organization name |
Cold Spring Harbor Laboratory
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| Department |
Delbruck Bldg.
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| Lab |
Martienssen
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| Street address |
1 Bungtown Rd
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| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
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| Platform ID |
GPL33489 |
| Series (2) |
| GSE234923 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference [RNA-seq]] |
| GSE234925 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference |
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| Relations |
| BioSample |
SAMN35739895 |
| SRA |
SRX20696762 |
| 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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