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Sample GSM4184016 Query DataSets for GSM4184016
Status Public on Dec 24, 2021
Title SLX_14255_i701_i517
Sample type SRA
Source name empty control
Organism blank sample
Characteristics reporter line: empty
possible cell type: None
developmental stage: empty control
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. For single cell transcriptomics, protoplasts were produced and isolated as described here, but sorted individually into 96-well plates.
Libraries were prepared using Illumina Nextera XT DNA preparation kit and either Nextera XT index kit – 96 indexes or Nextera XT index kit v2.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 4000
Data processing De-multiplexed fastq files were aligned using HISAT2
Picard SortSam was used to create .bam from .sam files, Picard Merge2 to merge .bam files where necessary
Cuffquant was used to create abundance files
Cuffnorm was used with all abundance files from all cells to get read counts.
Genome_build: TAIR10, Release 36
Supplementary_files_format_and_content: tab-delimited text file with read counts for each cells and gene
Submission date Nov 21, 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 GPL27787
Series (1)
GSE140778 Cellular trajectory analysis links tissue maturation to cellular specialization in the plant meristem
BioSample SAMN13342933
SRA SRX7197411

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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