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| Status |
Public on Jul 25, 2023 |
| Title |
Tpd pollen small RNA sterile rep2 |
| 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 |
For comparisons between Tpd1+/-;Tpd2+/-, Tpd1+/-;tpd2 and tpd1;tpd2 pollen, three biological replicates were used. Two biological replicates were used for dcl2T-/- and dcl2-mu1-/- pollen samples. 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.
Libraries were constructed with the NEXTFLEX Small RNA-Seq V3 kit (PerkinElmer, cat#NOVA-5132-06) using 2ug of total RNA input per library and the gel-free size selection protocol. 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 1x76bp run.
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
srna-tpd-pollen.sterile.22nt.pos.bw
srna-tpd-pollen.sterile.22nt.neg.bw
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| Data processing |
Adapters (5’-TGGAATTCTCGGGTGCCAAGG -3’) were trimmed using cutadapt (Marcel 2011) and the 4bp UMI sequences on either side of each read were removed.
Reads were filtered using pre-alignment to a maize structural RNA consensus database using bowtie2. Alignment and de novo identification of small RNA loci was performed with ShortStack. For any given biological comparison, reads from all samples were merged while simultaneously retaining sample-specific information. This allowed for the identification of a maximum number of putative small RNA loci (CPM≥5). Degradation productions are frequently mis-classified as sRNA producing loci. To avoid this, only clusters with clear size bias (21, 22, or 24nt) were retained in downstream analysis. This set of high-confidence sRNA annotations, along with edgeR, were used to determine differential sRNA accumulation between genotypes or tissues (log2FC ≥ 2, FDR ≤ 0.01). The accumulation of size and strand-biased hp-siRNAs was used to identify hairpin loci throughout the genome. For each locus, the underlying primary sequence was tested for reverse complementarity and RNA secondary structure prediction was performed using RNAfold. Non-hairpin siRNA targets were only retained if they showed negligible strand-bias (i.e., evidence of a dsRNA template for processing by a Dicer-like enzyme).
Assembly: Zm-W22-REFERENCE-NRGENE-2.0; Zm-TPD-1.0
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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) |
| GSE234924 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference [small RNA-seq] |
| GSE234925 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference |
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| Relations |
| BioSample |
SAMN35739910 |
| SRA |
SRX20696777 |
| 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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