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Sample GSM4744605

Query DataSets for GSM4744605

Status

Public on Nov 01, 2020

Title

H27471_heart_sciRNA

Sample type

SRA

Source name

RNA-seq of Homo sapiens: Fetus heart

Organism

Homo sapiens

Characteristics

development stage: Fetus tissues
tissue: Fetus heart

Growth protocol

Human fetal tissues were obtained by the UW Birth Defects Research Laboratory (BDRL) under a protocol approved by the University of Washington Institutional Review Board. Gestational age, reported as the number of weeks post-fertilization, was estimated from fetal foot length. Tissues of interest were isolated and rinsed in 1X HBSS (with Ca2+ and Mg2+) then blotted dry on a semi-damp gauze. Dried tissue was placed on a heavy-duty foil or in cryotube, snap frozen in liquid nitrogen, and then stored at -80°C.

Extracted molecule

polyA RNA

Extraction protocol

On the day of nuclei isolation, aliquots of tissue powder (0.1-1g) were first incubated with 1 mL ice-cold cell lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630 from (Buenrostro et al. 2013), modified to also include 1% SUPERase In (Thermo Fisher Scientific, AM2696) and 1% BSA (NEB, B9000S)) and then transferred to the top of a 40 µm cell strainer (VWR, 10199-654). Tissues were homogenized through the strainer with the rubber tip of a syringe plunger (VWR, BD309646) in 4 ml cell lysis buffer. The filtered nuclei were then transferred to a new 15 ml tube (VWR, 21008-936) and pelleted by centrifuge at 500xg for 5 min and washed once with 1 ml cell lysis buffer. The nuclei were fixed in 5 ml ice-cold 4% paraformaldehyde (EMS, 15-4-100) for 15 min on ice. After fixation, the nuclei were washed twice in 1 ml nuclei wash buffer (cell lysis buffer without IGEPAL), and re-suspended in 500 µl nuclei wash buffer. The samples were split into two tubes with 250 µl in each tube and flash frozen in liquid nitrogen. For human cell extraction in renal and digestive organs (kidney, pancreas, intestine, and stomach) and paraformaldehyde fixation, we followed the procedure described in (Thomsen et al. 2016).
sci-RNA-seq3 library: The paraformaldehyde fixed nuclei were processed similarly to the published sci-RNA-seq3 protocol (Cao et al. 2019). For paraformaldehyde fixed cells, frozen fixed cells were thawed on 37°C water bath, spun down at 500xg for 5 min, and incubated with 500ul PBSI (1 x PBS, pH 7.4, 1% BSA, 1% SuperRnaseIn) including 0.2% Triton X-100 for 3min on ice. Cells were pelleted and resuspended in 500ul nuclease free water including 1% SuperRnaseIn. 3ml 0.1N HCl were added into the cells for 5min incubation on ice (Rosenberg et al. 2018). 3.5ml Tris-HCl (pH = 8.0) and 35ul 10% Triton X-100 were added into cells to neutralize HCl. Cells were pelleted and washed with 1ml PBSR. Cells were pelleted and resuspended in 100ul PBSI. The following steps were similar with the sci-RNA-seq3 protocol (with paraformaldehyde fixed nuclei) with slight modifications: (1) We distributed 20,000 fixed cells (instead of 80,000 nuclei) per well for reverse transcription. (2) We replaced all nuclei wash buffer in following steps with PBSI. (3) All nuclei dilution buffer were replaced with PBS + 1% BSA.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Read alignment and gene count matrix generation for the single cell RNA-seq was performed using the pipeline that we developed for sci-RNA-seq3 (Cao et al. 2019) with minor modifications: Duplicates were removed using the unique molecular identifier (UMI) sequence (ED < 2, including insertions and deletions), reverse transcription (RT) index, hairpin ligation adaptor index and read 2 end-coordinate.
Genome_build: human reference genome (hg19)
Supplementary_files_format_and_content:
S1_metadata_cells.txt: Metadata of high-quality cells. Includes sample metadata and various per-cell QC stats, Louvain cluster id and cell type annotation, for each of the 4,062,965 high-quality cells used in the downstream analyses.
S2_Metadata_genes.txt: Metadata of genes. Includes gene id, short name and gene type information for each gene.
S3_gene_count.loom: Gene count matrix of cells. Includes expression UMI values for each gene in each cell. The gene count is stored in loom-formatted hdf5 file (read with function "scanpy.read_loom" in python)
S4_gene_expression_tissue.txt: Matrix of gene expression values across tissues. Includes normalized gene expression values (transcripts per million) for each tissue.
S5_gene_fraction_tissue.txt: Matrix of positive cell ratio for each gene across tissues. Includes the ratio number of cells expressing each gene in each tissue.
S6_gene_expression_celltype.txt: Matrix of positive cell ratio for each gene across tissues. Includes the ratio number of cells expressing each gene in each tissue.
S7_gene_fraction_celltype.txt: Matrix of positive cell ratio for each gene across main cell types. Includes the ratio number of cells expressing each gene for each type in each tissue.
S8_DE_gene_cells.txt: Differential gene expression test results for main cell types within each organ. For each gene, the “max_cell_type” is the cell type with the highest expression within the organ by transcripts per million (TPM) (“max.expr”). The “second_cell_type” is the cell type with the second highest expression within the organ by transcripts per million (TPM) (“second.expr”). The “fold.change” is the fold change between the max expression and second max expression. The “qval” is the false detection rate (one-sided likelihood ratio test with adjustment for multiple comparisons) for the differential gene expression test across different cell types within the organ.

Submission date

Aug 24, 2020

Last update date

Nov 01, 2020

Contact name

Junyue Cao

E-mail(s)

cao1025@uw.edu

Organization name

University of Washington

Department

Department of Genome Sciences