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Sample GSM4223960 Query DataSets for GSM4223960
Status Public on Dec 24, 2021
Title CD_2
Sample type SRA
 
Source name FACS-isolated protoplasts
Organism Arabidopsis thaliana
Characteristics age: 5day old seedlings
tissue: FACS-isolated protoplasts
line: pCVP2::DBOX-3YFP
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 5 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 which contains 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.
Extracted molecule total RNA
Extraction protocol Arabidopsis root protoplasts were isolated according to the published protocol (Clark et al., 2018). In short, sterilized seeds were plated onto the nylon mesh (57-103, Nitex) placed on the top of MS agar media. Two rows of seeds comprising approximately 100-150 seeds per row were then grown vertically for 5 days in long-day growth condition. To isolate root protoplasts, ⅓ of the root was cut and chopped on the surface of the mesh and collected in buffer containing cell wall degrading enzymes - 1.5 g Cellulase (C1794, Sigma) and 0.1 g Pectolyase (P3026, Sigma) in 100 ml protoplasting solution. After 1 hour of incubation with occasional stirring, the protoplasts were spun down (200 rcf for 6 min) in 15 ml Falcon tube and after removal of supernatant, cells were resuspended in 700 ul protoplasting solution without enzymes. After filtering with 70 um and 40 um cell strainers sequentially, FACS was performed using 70 micron nozzle at 10 psi. Fluorescence positive cells were collected in a round-bottom polystylene tube that contains 150 - 200 ul RLT buffer with beta-mercaptoethanol (10 ul / 1 ml RLT buffer, RNeasy micro kit protocol, Qiagen). Cell sorting was performed for about 15 min and collected cells were immediately frozen on dry ice and kept in -80 °C for up to one week.
Frozen cells were quickly thawed by adding the same volume of EtOH (150 - 200 µl), and RNA extraction was done using RNeasy Micro Kit (Qiagen) as previously described by Clark et al., 2018. RNA integrity was measured with Agilent HS RNA TapeStation system following the manufacturer’s instructions. Samples of RNA integrity value (RIN) 6.3 and above were taken to the following cDNA synthesis step. Clontech Smarter Ultra Low Input Library Kit V4 was used for cDNA synthesis and amplification (18 PCR cycles). Samples were then submitted to Novogene for library construction and RNA sequencing on an Illumina HiSeq SE50 run. The precise name of the instrument used to produce the reads is not available, although it was a HiSeq series instrument.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing  Raw counts have been aligned and mapped according to a standardized Bowtie 2 suit using the TAIR10 genome.
Feature Counts was used to obtain read counts
Differential gene expression analysis was performed using edgeR
Shannon-entropy based selection of tissue specific genes as described by Zambelli et al., 2018, using published root map RNA-seq data of other tissue types (Li S et al., 2016)
Genome_build: TAIR10
Supplementary_files_format_and_content: Tables with read counts, tab delimited text files.
 
Submission date Dec 18, 2019
Last update date Dec 24, 2021
Contact name Bernhard Blob
Organization name Sainsbury Laboratory, Cambridge University
Street address 47 Bateman Street
City Cambridge
ZIP/Postal code CB21LR
Country United Kingdom
 
Platform ID GPL17639
Series (1)
GSE142259 Cellular trajectory analysis links tissue maturation to cellular specialization in the plant meristem [protophloem sieve element cells]
Relations
BioSample SAMN13623609
SRA SRX7409475

Supplementary file Size Download File type/resource
GSM4223960_CD_2.txt.gz 129.1 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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