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Sample GSM4191811

Query DataSets for GSM4191811

Status

Public on Dec 24, 2021

Title

ZAT14_14h_1

Sample type

SRA

Source name

whole root meristems

Organism

Arabidopsis thaliana

Characteristics

age: 5day old seedlings
induction time: 14h Est
line: pWOL(XVE)::ZAT14-TURQ

Growth protocol

Arabidopsis seeds were surface sterilized in a bleach solution (5% sodium hypochlorite with 0.02% tween 20) for 5 minutes with gentle rotation. Seeds were then thoroughly washed at least 8 times with sterile distilled water and imbibed for 2 – 5 days at 4 °C in the dark. Seeds were germinated on 1/2 strength Murashige and Skoog (½ MS) agar medium containing 1% sucrose and 0.8% difco agar. Seedlings and adult plants were grown under long-day condition – 16 hours light (188 μmol m−2 s−1) and 8 hours dark at 23 °C. For confocal imaging, anatomical analysis, and RNA-seq experiments, 5-day-old vertically grown seedlings were used, unless otherwise specified.

Extracted molecule

total RNA

Extraction protocol

Col-0 seedlings carrying pWOL[xve]::ZAT14-mTurq and pWOL[xve]::ZAT14like-mTurq constructs were transferred to ½ MS plates supplemented with B-estradiol (5uM final concentration) 10 and 14 hours prior sampling the root meristems for RNA extraction. After induction, 5 days old seedlings were transferred to 1/2 MS medium plates with 5% agar in batches of 15 for easier dissection. Root meristems were cut off with a syringe needle and immediately transferred to RNAlater solution. All meristems were collected in total volume of 20ul of RNAlater. Total RNAs were extracted using RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. Library preparation and sequencing was done by Novogene (beijing, China) as follows: After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic samples, rRNA is removed using a specialized kit that leaves the mRNA. The mRNA from either eukaryotic or prokaryotic sources is then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library is ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

ZAT14like_ZAT14_OE_data_FPKM.txt
processed data header: Z14_1

Data processing

Analysis was performed by Novogene (Beijing, China). In short, raw reads are filtered to remove reads containing adapters or reads of low quality, so that downstream analyses are based on clean reads. The filtering process is as follows: (1) Discard reads with adaptor contamination. (2) Discard reads when uncertain nucleotides constitute more than 10 percents of either read (N > 10%). (3) Discard reads when low quality nucleotides (base quality less than 20) constitute more than 50 percents of the read. RNA-seq Adapter sequences (Oligonucleotide sequences of adapters from TruSeqTM RNA and DNA Sample Prep Kits): RNA 5' Adapter (RA5), part # 15013205: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' RNA 3' Adapter (RA3), part # 15013207: 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC(6-nucleotide index)ATCTCGTATGCCGTCTTCTGCTTG-3'. TopHat2 was used for aligment to TAIR10. The mismatch parameter is set to two, and other parameters are set to default. Appropriate parameters are also set, such as the longest intron length. Only filtered reads are used to analyze the mapping status of RNA-seq data to the reference genome. the gene expression level is estimated by counting the reads that map to genes or exons. HTSeq software was used to analyze the gene expression levels in this experiment, using the union mode. The result files present the number of genes with different expression levels and the expression level of single genes. In general, an FPKM value of 0.1 or 1 is set as the threshold for determining whether the gene is expressed or not.
Genome_build: TAIR10
Supplementary_files_format_and_content: read counts or FPKM values, as tab delimeted tables in .txt files

Submission date

Nov 25, 2019

Last update date

Dec 24, 2021

Contact name

Bernhard Blob

Organization name

Sainsbury Laboratory, Cambridge University

Street address

47 Bateman Street

City

Cambridge