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| Status |
Public on Jul 25, 2023 |
| Title |
Tpd pollen iPARE fertile 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 |
polyA 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.
For iPARE-seq libraries, 40ug of total RNA was poly(A) selected using a Dynabeads mRNA Purification Kit (ThermoFisher, cat#61006). 1ug of poly(A) RNA was ligated to the 5’ PARE adapter (100pmol) in 10% DMSO, 1mM ATP, 1X T4 RNA ligase 1 buffer (New England Biolabs, cat#B0216L), 25% PEG8000 with 1ul (40U) of RNaseOUT (ThermoFisher, cat#10777019) and 1ul T4 RNA ligase 1 (New England Biolabs, cat#M0204S) in a reaction volume of 100ul. Ligation reactions were performed for 2 hours at 25˚C followed by overnight incubation at 16˚C. Samples were then purified using RNA Clean XP beads (Beckman Coulter, cat#A63987) and eluted in 18ul dH20. Chemical fragmentation of ligated RNA to ≤200nt was performed using the Magnesium RNA fragmentation kit (New England Biolabs, cat#E6150S). 2ul RNA Fragmentation Buffer was added and samples were incubated at 94˚C for 5 minutes followed by a transfer to ice and the addition of 2ul of RNA Stop solution. Samples were purified using the RNA Clean & Concentrator-5 kit (Zymo Research, cat#R1013) and eluted in 11ul H20. Reverse-transcription was performed as follows: 10ul of RNA, 1ul 10mM dNTP, and 2ul random primer mix (New England Biolabs, cat#S1330S) were mixed and incubated for 10 minutes at 23˚C then put on ice for 1 minute. The following was then added: 4ul 5X SuperScript IV buffer, 1ul 100mM DTT, 1ul RNaseOUT, and 1ul Superscript IV (200U). The reaction was incubated for 10 minutes at 23˚C followed by 10 minutes at 50˚C. 80ul of TE was then added to this mixture. Target indirect capture was performed with 100ul Dynabeads MyOne Streptavidin T1 beads (ThermoFisher, cat#65601) as per manufacturer instructions. 100ul of the RT reaction was used as input, and captured cDNA molecules were eluted in 50ul. Second-strand synthesis was performed using 5U Klenow fragment (New England Biolabs, cat#M0210S) with 100uM dNTPs and 1uM of iPARE adapter primer (5’-NNNNTCTAGAATGCATGGGCCCTCCAAG-3’) for 1 hour at 37˚C, incubation at 75˚C for 20 minutes. Samples were purified with a 1:1 ratio of AMPure XP SPRI beads (Beckman Coulter – cat#A63880) and resuspended in 51ul EB. 50ul of sample was used for library preparation with the NEB Ultra DNA library kit (New England Biolabs, cat# E7370S). Barcoded samples were sequenced with a NextSeq500 2x150bp high output run. Use of the directional iPARE adapter allows for the retention of directionality even when using a non-directional DNA-seq kit.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ipare-fertile.norm.bdg.gz
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| Data processing |
library strategy: iPARE-seq
Cutadapt (Marcel 2011) was used to search and recover the adapter sequence in both 5’ and 3’ orientation (forward in read1 or read2 respectively). Read1 adapter reads were trimmed for the 3’ adapter if present, and the 5’ iPARE adapter was subsequently removed. Potential polyA tails were also removed, and only reads ≥20nt were retained. Read2 adapter reads were processed in an identical fashion. Filtered reads were mapped using Bowtie2 (Langmead and Salzberg 2012) and the 5’ position of each read (the cloned 5’ monophosphate) was extracted using BEDtools (Quinlan and Hall 2010) with CPM normalization. Small RNA target prediction was performed using psRNATarget (Dai et al. 2018).
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) |
| GSE234922 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference [iPARE-seq] |
| GSE234925 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference |
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| Relations |
| BioSample |
SAMN35738779 |
| SRA |
SRX20696757 |
| 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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