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Sample GSM3511858

Query DataSets for GSM3511858

Status

Public on Mar 25, 2019

Title

WT rep1

Sample type

SRA

Source name

Root

Organism

Arabidopsis thaliana

Characteristics

strain/background: Col-0
genotype/variation: Wild type
tissue: Root
age: 6-day-old seedlings
condition: Protoplasted

Treatment protocol

For protoplasting: Roots of 6-day-old seedlings were cut approximately one centimetre from their tip, broadly diced with a scalpel blade, and treated with 7 ml protoplasting solution optimised for scRNA-seq from a protocol in Birnbaum et al., 2003. Immediately before use, 1.5% Cellulase R-10 and 0.1% Pectolyase (Duchefa Biochem.) were added to fresh protoplast buffer (0.1 M KCl, 0.02 M MgCl2, 0.02 M CaCl2, 0.1% BSA (Sigma Aldrich)), 0.08 M MES, and 0.6 M Mannitol adjusted to pH 5.5 with 0.1M Tris HCl) and mixed thoroughly. Root tissues were protoplasted for 2 hours at 20°C on an orbital shaker set at 200 evolutions/minute. The mixture was subsequently filtered through a 100 μm nylon filter and rinsed with 1-5 ml of root protoplast buffer. Protoplasts were then centrifuged for 10 minutes (500 g – 4°C), the supernatant gently removed, and the pellet resuspended in 10 ml root protoplast buffer containing 0.4 M Mannitol and no CaCl2. This wash procedure was repeated once more, the protoplasts centrifuged as before, and resuspended in ~500 μl or less protoplast buffer without CaCl2 and with 0.4 M Mannitol. Protoplasts were validated under a light microscope, and if necessary any excess debris or un-protoplasted tissues removed with an additional washing step. Cells were filtered through a 40 μm cell strainer (Flowmi Bel Art SP Scienceware), quantified using a haemocytometer, and adjusted to a density of approximately 800-‐900 cells per μl.

Growth protocol

Seedlings were grown vertically on nylon mesh on ½ Murashige and Skoog 1% agar plates.

Extracted molecule

total RNA

Extraction protocol

RNA extraction (for bulk RNA-seq samples) - either protoplasted (reduced to smallest possible volume) or unprotoplasted root tissue was flash-frozen in liquid nitrogen, ground to a powder and RNA extracted using the Spectrum Plant RNA Extraction Kit (Sigma). Quality and quantity were checked using Agilent Bioanalyzer chips and Nanodrop, respectively, prior to library construction.
Bulk RNA libraries were produced using the NEBNext Poly(A) mRNA Magneticb Isolation Module, and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with NEB Multiplex oligos. Final library size and quality were checked on a DNA High Sensitivity Bioanalyzer chip (Agilent), and libraries were quantified using the NEBNext Library Quantification Kit for Illumina. Single cell RNA-seq libraries were prepared from fresh protoplasts according to the 10x Genomics Single Cell 3’ Reagent Kit v2 protocol. For each replicate, cells were loaded in the 10x Genomics Chromium single cell microfluidics device with the aim of capturing 7,000 cells. 11 cycles were used for cDNA amplification, as well as for final PCR amplification of the adapter-‐ligated libraries. Final library size and quality were checked on a DNA High Sensitivity Bioanalyzer chip (Agilent), and libraries were quantified using the NEBNext Library Quantification Kit for Illumina.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

Single cell RNA-seq libraries were prepared from fresh wild-type root protoplasts according to the 10x Genomics Single Cell 3’ Reagent Kit v2 protocol. For each replicate, 12,200 cells were loaded in the 10x Genomics Chromium single cell microfluidics device with the aim of capturing 7,000 cells. 11 cycles were used for cDNA amplification, as well as for final PCR amplification of the adapter-‐ligated libraries. Final library size and quality were checked on a DNA High Sensitivity Bioanalyzer chip (Agilent), and libraries were quantified using the NEBNext Library Quantification Kit for Illumina.
The R1 raw data files include the technical reads; the first 16 basepairs are barcode and the last 10 basepairs are UMI. The R2 raw data files include the biological reads.
processed data file: Root_single_cell_wt_datamatrix.csv

Data processing

Cell Ranger 2.0.2 (10X Genomics) was used to process scRNA-seq data.
Bulk RNA Sequencing reads (pair-end, 75 bp) were trimmed using Trimmomatic (version 0.36), and aligned to the Arabidopsis TAIR10 reference genome with STAR. Gene expression values were calculated on uniquely mapped reads using HTSeq (version 0.7.2).
Genome_build: TAIR10
Supplementary_files_format_and_content: *_datamatrix.csv: Comma-delimited text files include UMI counts values in each cell.
Supplementary_files_format_and_content: Root_bulk_tissue_datamatrix.txt: Tab-delimited text file includes read counts and RPM values for each Sample.

Submission date

Dec 13, 2018

Last update date

Jun 18, 2020

Contact name

Marja Timmermans

Organization name

University of Tuebingen

Street address

Morgenstelle 32

City

Tuebingen