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Sample GSM7919274

Query DataSets for GSM7919274

Status

Public on Nov 28, 2023

Title

Col-0 low-light rep5

Sample type

SRA

Source name

whole flowers, pre-mitosis I anthers

Organism

Arabidopsis thaliana

Characteristics

tissue: whole flowers, pre-mitosis I anthers
genotype: Col-0
treatment: low light (50umol/m2/s)

Treatment protocol

At the onset of flowering, just before bolting, treatments were commenced. In normal light conditions, plants were illuminated with a Photosynthetic Photon Flux Density (PPFD) of 205(± 8.6 SD) μmol/m2/s, while the low light plants received 53 (± 5) μmol/m2/s PPFD through use of net shading. Trays were rotated weekly to minimise localised environmental effects.

Growth protocol

Arabidopsis thaliana Columbia (Col-0) ecotype and bhlh89,91 (SALK-123106 x GK-345C06) T-DNA mutants were sown onto a 3:1 mix of Levington M3 compost: Vermiculite with T34 biological control. Plants were grown at 22°C in a controlled environment room with 16h day length provided by fluorescent lighting.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from pre-mitotic buds, containing anther stages 1-10, from 4 factor groups (bhlh89,91 Low, bhlh89,91 Norm, Col-0 Low, Col-0 Norm), each with 4 biological replicates, using RNeasy Kit (Qiagen)
Poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. mRNA was fragmented into small pieces using divalent cations under elevated temperature. Cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. A single 'A' base was then added to cDNA fragments and adapter was ligated. Products were then purified and enriched with PCR amplification. PCR yield was quantified by Qubit and samples were pooled to make single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and pair-end reads of 100 bp were read through on the BGISEQ-500 platform, combining the DNB nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Description

Col-Low-5

Data processing

BGI’s internal software SOAPnuke was used to filter reads: removing reads with adaptors, reads in which unknown bases (N) are more than 1%, and removing low quality reads (percentage of bases with less than 15% quality more than 40%). Clean reads were mapped to Arabidopsis thaliana Reference Transcript Dataset 2 (AtRTD2) using Salmon then read counts and transcript per million reads (TPMs) were generated using tximport R package version 1.10.0 and length Scaled TPM method.
Assembly: AtRTD2
Supplementary files format and content: CSV file containing TPMs per gene
Supplementary files format and content: CSV file containing gene read counts

Submission date

Nov 27, 2023

Last update date

Nov 28, 2023

Contact name

Zoe Wilson

Organization name

University of Nottingham

Department

Plant Sciences

Street address

Sutton Bonington Campus