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Sample GSM5773905

Query DataSets for GSM5773905

Status

Public on Jan 09, 2022

Title

protoplasted rep1

Sample type

SRA

Source name

Tobacco BY-2 cell line

Organism

Nicotiana tabacum

Characteristics

cell line: BY-2
age: three day-old BY-2 suspension cells
condition: protoplasted

Treatment protocol

Protoplasts were prepared from 3 d-old tobacco BY-2 suspension cells. The 40-ml cell culture was filtered using a 40-μm nylon mesh (BD Falcon, USA) was to collect BY2 cells, which were washed twice with protoplast wash solution (0.5 M mannitol, 4 mM 2-(N-morpholino) ethanesulfonic acid (pH 5.7)). The cells were then divided into three parts. One part was immediately cryopreserved in liquid nitrogen for bulk RNA paired-end sequencing (with three replicates), the other two parts were resuspended in 10 mL of enzyme solution (pH 5.7), containing 1.5% cellulase "Onozuka RS" (Yakult Pharmaceutical Ind. Co. Ltd., Tokyo, Japan), 0.5% macerase (Yakult Pharmaceutical Ind. Co. Ltd., Tokyo, Japan), 0.5 M mannitol, and 4 mM 2-(N-morpholino) ethanesulfonic acid , in a 20-ml Petri dish and incubated for 2 h at 26°C on a rotary shaker that is maintained in the dark at 60 rpm. Protoplasts were recovered by filtration through a 40-μm nylon mesh and washed twice with protoplast wash solution at 150 g for 3 min.

Growth protocol

Tobacco (Nicotiana tabacum) BY-2 (Bright Yellow-2) cells were obtained from Miu’s laboratory (School of Biological Engineering in Huainan Normal University, Anhui, China) and cultured in Murashige and Skoog medium (sigma, M5524, USA), pH 5.8, with the following supplements: 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g/L sucrose, 200 mg/L KH2PO4, 100 mg/L myo-inositol and 1 mg/L thiamin (VB1). BY2 cells were grown in 40 ml culture medium in 200 ml Erlenmeyer flasks on a rotary shaker that is maintained in the dark at 150 rpm and 26°C. BY-2 cells were sub-cultured weekly from a 7 d-old liquid culture by transferring 5 mL BY-2 cells to 40 mL fresh BY-2 culture medium.

Extracted molecule

polyA RNA

Extraction protocol

The obtained BY2 protoplasts were divided into two parts and cryopreserved, one for bulk RNA paired-end sequencing (with three replicates) and another for single-cell RNA sequencing (with two replicates). Illumina libraries were constructed with Gene Expression v3 kits (10X Genomics).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

BY5_bulk_datamatrix.txt

Data processing

The raw data were converted into fastq files by using fastq-dump (version 2.9.6). After demultiplexed, raw reads were mapped to the reference genome (K326) using the 10X Genomics CellRanger (version 4.0.0) pipeline with the default parameters.
The reference genome of the tobacco BY2 cell line is current unavailable, we thus choose the K326 genome of Nicotiana tabacum to align the bulk RNA-seq reads) with STAR (Dobin et al., 2013). The K326 genome was downloaded from the SOL genomics database (https://solgenomics.net/) (Sierro et al., 2014). The read alignment rates of the six samples were 90.54% - 91.84%, indicating that the K326 reference genome can be used for BY2 transcriptome data quantification. Gene expression values were calculated based on the uniquely mapped reads using featureCounts (version 2.0.2; Liao et al., 2014), and DEseq2 (version 1.28.1; Love et al., 2014) was used to identify differentially expressed genes.
Genome_build: K326_2014
Supplementary_files_format_and_content: *_datamatrix.csv: Comma-delimited text files include UMI counts values in each cell.

Submission date

Jan 06, 2022

Last update date

Jan 09, 2022

Contact name

Yao Jie

E-mail(s)

andy-yaoj@zju.edu.cn

Phone

15158110186

Organization name

ZheJiang University