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Sample GSM7906394

Query DataSets for GSM7906394

Status

Public on Nov 18, 2023

Title

Mock, RNA, rep2

Sample type

SRA

Source name

Leaf

Organism

Arabidopsis thaliana

Characteristics

tissue: Leaf
cultivar: Col-0
treatment: Mock

Extracted molecule

total RNA

Extraction protocol

Fresh Nuclei purification buffer (NPB; 15mM Tris pH 7.5, 2mM EDTA, 80mM KCl, 20mM NaCl, 0.5mM spermidine, 0.2mM spermine, 1:100 BSA, and 1:100 protease inhibitor cocktail) was prepared before the experiment and chilled on ice. All the following procedures were performed on ice or at 4ºC. Twenty leaves were chopped in 500-1,000 µL of cold NPB with 1:500 Protector RNase IN with a razor blade on ice for 5 min to release nuclei and incubated in 20 mL of NPB. The crude nuclei extract was sequentially filtered through 70 µm and 30 µm cell strainers. Triton-X and NP40 were added to the extract at the final concentration of 0.1% each, and the extract was incubated at 4ºC for 5 min with rotation. The nuclei suspension was centrifuged at 50 x g for 3 min in a swing-rotor centrifuge to pellet non-nuclei debris, and the supernatant was taken. Nuclei were pelleted by centrifugation at 500 x g for 5 min with a swing-rotor centrifuge. When the pellet was green, the pellet was resuspended in 20 mL of NPBd (NPB, 0.1% Triton-X, and 0.1% NP40) with 1:1000 Protector RNase IN by pipetting, followed by centrifugation at 700 x g for 5 min. When the pellet was translucent, this step was skipped. The pellet was then washed by resuspending it in 20 mL of NPB with 1:1000 Protector RNase IN and centrifuging at 500 x g for 5 min with a swing-rotor centrifuge. The pellet was resuspended in 950 µL of 1X Nuclei Buffer (10X Genomics; PN-2000207) with 1:20 Protector RNase IN and 1mM DTT. The nuclei suspension was centrifuged at 50 x g for 3 min in a swing-rotor centrifuge to pellet non-nuclei debris, and the supernatant was taken. This step was repeated one more time. Then, nuclei were counted manually with a hemocytometer. Nuclei were pelleted by centrifugation at 500 x g for 5 min with a swing-rotor centrifuge and removed supernatant leaving approximately 10 µL of buffer. Nuclei were counted again, and up to 16k nuclei were used for the following steps. Single-cell RNA-seq and ATAC-seq libraries were constructed according to the manufacturer’s instruction (10X Genomics, catalog: CG000338). Single-nucleus RNA-seq libraries were sequenced with Illumina NovaSeq 6000 in dual-indexmode with 10 cycles for i7 and i5 index. Single-nucleus ATAC-seq libraries were also sequenced with Illumina NovaSeq 6000 in dual-indexmode with eight and 24 cycles for i7 and i5 index, respectively.
We followed the 10X genomics multiome assay protocols

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

10x Genomics Multiome
combined_filtered.rds

Data processing

Sequence data were processed to obtain single cell feature counts by running cellranger (v6.0.1) and cellranger-arc (v2.0.0) for snRNA-seq and snATAC-seq, respectively. For snRNA-seq, the –include-introns option was used to align reads to the A. thaliana nuclear transcriptome built with the TAIR10 genome and Araport 11 transcriptome. Count data were analyzed with the R packages Seurat v4 (Hao et al. 2021) and Signac (Stuart et al. 2021).
QC matrices for snATAC-seq were generated by a modified version of loadBEDandGenomeData function in the R package Socrates. ACRs were identified with MACS2 (Zhang et al. 2008) with the following parameters: -g (genomesize)=0.8e8, shift=-50, extsize=100, and --qvalue=0.05, --nomodel, --keep-dup all. Fraction of reads mapping to within 2kb upstrem or 1kb downstream TSS was calculated. Nuclei were filtered with the following criteria: 200 < RNA UMI count < 7000, RNA gene count > 180, 200 < ATAC UMI count <20000, fraction of RNA reads mapped to mitochondrial genome < 5%. Seurat objects of individual samples were merged with the Merge_Seurat_List function of the scCustomize package.
Supplementary files format and content: combined_filtered.rds contains snRNA-seq and snATAC-seq data from all samples after cell filtering
Supplementary files format and content: Processed files except for combined_filtered.rds contains snRNA-seq and snATAC-seq data of each sample before cell filtering

Submission date

Nov 17, 2023

Last update date

Nov 19, 2023

Contact name

Joseph R Ecker

E-mail(s)

ecker@salk.edu

Phone

8584534100

Organization name

HHMI-Salk-Institute