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Sample GSM8587467

Query DataSets for GSM8587467

Status

Public on Oct 28, 2024

Title

AV1b, Atrioventricular node sample from av_1, replicate b

Sample type

SRA

Source name

Atrioventricular node

Organism

Rattus norvegicus

Characteristics

tissue: Atrioventricular node
individual or_pool: av_1
developmental stage: 17 week
Sex: male
strain: Wistar
disease: healthy

Treatment protocol

17-week-old healthy male Wistar rats

Extracted molecule

total RNA

Extraction protocol

17-week-old animals were euthanized between 10am and 12pm using CO2, followed by perfusion with PBS to remove excess blood. Right ventricle, left ventricle and septum were immediately rinsed with ice cold PBS, then minced on ice by sterile razor blade, finally mixed with 4mL of nuclei isolation buffer (NIB). Left atria, right atria, SA node, AV node, aorta (from aortic root to iliac bifurcation), pulmonary vein, and pulmonary artery were immediately frozen in LN2 and stored at -80°C until use. Since AV nodes from rat have an approximate volume of 0.5mm3, we pooled 3-4 rat samples to obtain 1 sample for further processing. The same approach was used for SA node.
Frozen tissues were mounted on OCT and sectioned at 60μm at -20°C using a cryotome (Leica CM 1950). Tissue slices were transferred to 4mL of ice-cold NIB (Hepes 20 mM, Sucrose 0.25M, MgCl2 3mM, KCl 25mM, Igepal-630 0.01%, BSA 0.5%, pH 7.2). All tissue mixtures were dounce homogenized on ice with 10 strokes of a loose fit pestle followed by 10 minutes incubation, followed by 10 strokes of a tight fit pestle and 10 min incubation. Homogenate was transferred to a 15mL conical tube, filled to 8mL with nuclei wash buffer (NWB = NIB without detergent), and centrifuged at 40g x 4’, at 4°C (Beckman Coulter Allegra X-15R swinging bucket centrifuge). Supernatant was filtered through sequential 40μm and 10μm meshes (Pluriselect, Germany) in 50mL conical tube, filtrate topped up to 10mL with NWB and centrifuged at 600g x 5’, at 4°C. Supernatant was discarded and pellet resuspended, transferred to 15mL tube, topped up with 8mL NWB centrifuged (600g x 5’, 4°C). Final pellet was resuspended in 150μL nuclei resuspension buffer (NRB = NWB with 1:80 murine RNAse inhibitor, NEB). All procedures were performed on ice. Nuclei, stained with Trypan blue, were manually counted using a hemocytometer (inCyto.com). 7,000 nuclei input (5,000 calculated recovery) per sample were used for droplet generation and library construction according to the manufacturer's protocol (10x Genomics, single-cell 3-prime V2 chemistry), with minor modifications. First, after emulsion generation, nuclei were incubated on ice for 15 minutes to promote nuclear lysis. Second, the reverse transcription protocol was extended to enable the retention of longer transcripts. Libraries were multiplexed at an average of 4-5 libraries per flow cell. Sequencing was performed on an Illumina Nextseq550 in the Broad Institute’s Genomics Platform (genomics.broadinstitute.org).

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

NextSeq 550

Description

10x Genomics 3' v2 chemistry

Data processing

CellRanger v3.0.2 was used to run cellranger mkfastq in order to generate FASTQ files.
Assembly: rn6
Supplementary files format and content: Creation of the h5 count matrix files for each sample included the following: (1) FASTQ trimming using cutadapt. (2) Cellranger count was used to obtain h5 count matrices using a GTF file from our manuscript (supplementary file rn6.broad.cardiac.3.gtf.gz here). Note that these h5 files have not had CellBender run on them.

Submission date

Oct 23, 2024

Last update date

Oct 28, 2024

Contact name

Stephen Jordan Fleming

E-mail(s)

[email protected]

Organization name

Broad Institute

Lab

Lab of Patrick Ellinor