tissue: Pectoralis major muscle age: 47 days posthatch Sex: Male wb status: WB-Unaffected chicken type: Crossbred BCD hatch: FBTG8 live wt (kg) day 29: 1.385 live wt (kg) day 46: 3 feed consumption (kg) day 29-46: 3.385 bird plant wt (kg): 3.045 breast muscle wt (kg): 0.65 abdominal fat wt (g): 56.2 p. major phu: 5.97
Treatment protocol
Samples were smashed into pieces in frozen state by hammering. Pulverized tissues were stored at -80°C until RNA extraction.
Growth protocol
Animal experiments for crossbred (BCD) and purebred chickens (line B and line C) are described in studies by Zhou et al. (Zhou, N., Lee, W.R. and Abasht, B., 2015. Messenger RNA sequencing and pathway analysis provide novel insights into the biological basis of chickens’ feed efficiency. BMC genomics, 16(1), p.195.) and Mutryn et al. (Mutryn, M.F., Brannick, E.M., Fu, W., Lee, W.R. and Abasht, B., 2015. Characterization of a novel chicken muscle disorder through differential gene expression and pathway analysis using RNA-sequencing. BMC genomics, 16(1), p.399.), respectively.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the tissue samples using the mirVanaTM miRNA Isolation Kit according to the manufacturer’s protocol (Life Technologies®).
Label
fluorescent tag barcodes
Label protocol
Please see “CodeSet Hybridization Setup” document
Hybridization protocol
Please see “CodeSet Hybridization Setup” document
Scan protocol
Please see “nCounter® Analysis System User Manual”
Description
Pectoralis major muscle_Crossbred BCD_WB-Unaffected
Data processing
For normalization of gene expression, negative control probes were used for detection of “background counts” in each hybridization reaction, and the medians of background counts were applied to background subtraction (NanoString User Manual, 2012). Next, 12 designated housekeeping genes were used for normalization of gene expression (nSolver software package; version 2.5). To prevent saturation issues that may occur due to 2 of the genes (GAPDH and MYH1E) that are highly expressed in muscle tissue, these 2 genes have been attenuated to 10% raw count levels for the samples that were proceed on 7/18/2014 (see the “Expression Assay Data” column). So, the normalized count levels for these two genes for these samples were divided by “0.1” to make them comparable with the values of prior processed samples on 12/05/2013 and 12/06/2013. Note that no attenuation was performed on GAPDH and MYH1E or any other genes for samples processed on 12/05/2013 or 12/06/2013.
