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Sample GSM6754837

Query DataSets for GSM6754837

Status

Public on Sep 21, 2023

Title

Bill skin Replicate 6

Sample type

SRA

Source name

Bill skin

Organism

Anas platyrhynchos

Characteristics

tissue: Bill skin

Growth protocol

Meissner corpuscles were manually collected and pooled for subsequent transcriptomic analysis. These experiments were conducted in RNAse-free conditions. An aspiration pipette was pulled from capillary glass tubing (G150F-3, Warner Instruments, Hamden, CT) using a micropipette puller (P-1000, Sutter, Novato, CA) with a tip diameter of ~40–60 µM, filled with 3 µl of the RNA Lysis Buffer (Quick-RNA Microprep Kit, Zymo, Irvine, Ca). The tip was loaded into micromanipulator and used to aspirate 2–10 corpuscles per pipette (per sample) by applying negative pressure using a high-speed pressure clamp system (HSPC-1, ALA Scientific Instruments). Collected corpuscles were then deposited into a 0.5 ml tube containing 10 µl of the RNA Lysis Buffer. Nearby skin cells absent of any corpuscles were also collected for comparison. Samples were then stored at −80°C until RNA isolation. 5-6 total replicates were collected from 3 independent skin isolations.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Quick-RNA Microprep Kit (Zymo) according to manufacturer’s instructions. RNA concentration and integrity number (RIN) were assessed by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). RNA concentrations for corpuscles were in the range of 63–323 pg/µl and RIN values were in the range of 6.1–8.0.
Sequencing libraries were prepared using the Kapa mRNA Hyper Prep kit (KAPA Biosystems, Wilmington, MA) (skin samples) or the NEBNext Single Cell/Low Input RNA Library Prep Kit (New England Biolabs, Ipswich, MA) (Meissner corpuscles samples).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

library kit used: Kapa mRNA Hyper Prep kit

Data processing

Primary analysis performed using Illumina's CASAVA 1.8.2 software suite.
Raw sequencing reads were filtered and trimmed to retain high-quality reads using Trimmomatic v0.39 (Bolger et al., 2014) with default parameters.
Filtered high-quality reads from all samples were aligned to duck reference genome using STAR aligner v2.7.7a with default parameters (Dobin et al., 2013).
Aligned reads were counted by the featureCounts module within the Subread package v2.0.1 with default parameters (Liao et al., 2014).
Processing, normalization, and statistical analysis of read counts was performed using the EdgeR v3.38.4 package in R v4.2.1 (Robinson et al., 2010)
Normalized read counts were further normalized to gene length and expressed as “fragments per kilobase gene length per million mapped reads” (FPKM).
Statistical analysis of differential expression of genes between groups was performed using the GLM approach and the quasi-likelihood F-test, as implemented in the EdgeR package.
Assembly: The duck reference genome and the gene annotation were obtained from the National Center for Biotechnology Information (Anas platyrhynchos; assembly ZJU1.0; NCBI Annotation Release: 104; all files accessed on 4/30/2021). Reference genome: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/015/476/345/GCF_015476345.1_ZJU1.0/GCF_015476345.1_ZJU1.0_genomic.fna.gz; Gene annotation: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/015/476/345/GCF_015476345.1_ZJU1.0/GCF_015476345.1_ZJU1.0_genomic.gff.gz; the gene annotation was filtered to include only protein-coding genes.
Supplementary files format and content: Processed data file in Excel format (.xlsx) contains raw read counts, normalized read counts, and FPKM values for 5/6 replicates/samples of Meissner corpuscles and bill skin for all protein-coding genes in duck genome annotation. It also includes mean normalized counts and FPKM values for both groups, P-values, adjusted FDR values, and fold change (log2) for statistical comparison of expression values between 2 groups.

Submission date

Nov 23, 2022

Last update date

Sep 21, 2023

Contact name

Viktor Feketa

E-mail(s)

viktor.feketa@yale.edu

Organization name

Yale University School of Medicine

Department

Cellular & Molecular Physiology