GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM8341842

Query DataSets for GSM8341842

Status

Public on Jul 05, 2024

Title

scATAC-seq_Leaf.rep2

Sample type

SRA

Source name

leaf

Organism

Glycine max

Characteristics

tissue: leaf

Extracted molecule

genomic DNA

Extraction protocol

Briefly, the tissue was finely chopped on ice for approximately 2 minutes using 600 μL of pre-chilled Nuclei Isolation Buffer (NIB: 10 mM MES-KOH at pH 5.4, 10 mM NaCl, 250 mM sucrose, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT, 1% BSA, and 0.5% Triton X-100). After chopping, the mixture was passed through a 40-μm cell strainer and centrifuged at 500 rcf for 5 minutes at 4°C. The supernatant was carefully decanted, and the pellet was reconstituted in 500 μL of NIB wash buffer (10 mM MES-KOH at pH 5.4, 10 mM NaCl, 250 mM sucrose, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT, and 1% BSA). The sample was filtered through a 10-μm cell strainer and gently layered onto 1 mL of 35% Percoll buffer (35% Percoll mixed with 65% NIB wash buffer) in a 1.5-mL centrifuge tube. The nuclei were centrifuged at 500 rcf for 10 minutes at 4°C. After centrifugation, the supernatant was carefully removed, and the pellets were resuspended in 10 μL of diluted nuclei buffer (DNB, 10X Genomics Cat# 2000207). Approximately 5 μL of nuclei were diluted tenfold, stained with DAPI (Sigma Cat. D9542), and the nuclei quality and density were evaluated using a hemocytometer under a microscope. The original nuclei were then diluted with DNB buffer to a final concentration of 3,200 nuclei per μL. Finally, 5 μL of nuclei (16,000 nuclei in total) were used as input for scATAC-seq library preparation.
scATAC-seq libraries were prepared using the Chromium scATAC v1.1 (Next GEM) kit from 10X Genomics (Cat# 1000175), following the manufacturer's instructions (10x Genomics, CG000209_Chromium_NextGEM_SingleCell_ATAC_ReagentKits_v1.1_UserGuide_RevE). Libraries were sequenced on an Illumina NovaSeq 6000 in dual-index mode with eight and 16 cycles for i7 and i5 indexes, respectively.

Library strategy

ATAC-seq

Library source

genomic single cell

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

Leaf.soc.processed.rds
Gm_atlas_Leaf_metav3_gene_sparse.rds
Gm_atlas_Leaf_ACR_4cpm_rmcds_ACR_sparse.rds
Gm_atlas_Leaf_ACR_4cpm_cellstates_counts.txt.gz

Data processing

The R package Socrates was used for nuclei identification and quality control.23 The BED file containing single base-pair Tn5 integration sites was imported into Socrates along with the soybean GFF gene annotation (Phytozome, version Gmax_508_Wm82.a4.v1) and the genome index file. To identify bulk-scale ACRs in Socrates, the callACRs function was employed with the following parameters: genome size=8.0e8, shift=-75, extsize=150, and FDR=0.1. This step allowed us to estimate the fraction of Tn5 integration sites located within ACRs for each nucleus. Metadata for each nucleus were collected using the buildMetaData function, with a TSS (Transcription Start Site) window size of 1 kb (tss.window=1000). Sparse matrices were then generated with the generateMatrix function, using a window size of 500. High-quality nuclei were identified based on the following criteria: a minimum of 1,000 Tn5 insertion sites per nucleus, at least 20% of Tn5 insertions within 2 kb of TSSs, and at least 20% of Tn5 insertions within ACRs across all datasets. Additionally, a maximum of 20% of Tn5 insertions in organelle genomes was allowed.
Assembly: Soybean William 82 v4 reference genome
Supplementary files format and content: Sorate object for clustering, gene accessibility matrix, ACR accessibility matrix, ACR count matrix for celltypes

Submission date

Jun 20, 2024

Last update date

Jul 05, 2024

Contact name

Robert J. Schmitz

E-mail(s)

[email protected]

Organization name

University of Georgia

Department

Genetics