Ethical statement
All experimental animals were handled according to a protocol approved by the Medical Ethics Committee of the First Affiliated Hospital, Medical College, Shihezi University (A2018-068-01). The methods employed in this research were performed in strict adherence to the approved guidelines. And this study is reported in accordance with ARRIVE guidelines (https:// arriveguidelines.org).
Immunohistochemical
A total of 100 fertilized chicken eggs from the RC and CB strains were incubated at 37 degrees Celsius and 60% humidity. The leg muscle tissues were extracted from the 8th (E8) and 15th (E15) day embryos of the RC chicken and CB, fixed in a 10% formaldehyde solution for 48 hours, and then stored in 70% alcohol. After treatment, leg muscle tissue samples were cut into 1 cm by 1 cm by 0.5 cm pieces, rendered transparent with wax, imbedded, sliced (to a thickness of 5μm), and baked. The diluted monoclonal anti-skeletal myosin (Fast/Slow) primary antibody (Sigma, Darmstadt, Germany) was incubated at 4°C overnight following dewaxing and antigen repair on paraffin sections, and the enzyme-conjugated goat anti-mouse IgG secondary antibody (ZSGB-Bio, Beijing, China) was incubated at 37°C for an hour. The tablets were sealed with neutral gum following DAB colour processing before being observed using a microscope image (40×10).
Sample preparation
The leg muscles from E8 and E15 were surgically removed and stored at -80°C after being instantly frozen in liquid nitrogen. The embryonic brain was collected for sex identification by referencing the protocol of Vucicevi14. Three replicates were set up for each cohort and female samples were chosen for high-throughput sequencing. After sequencing, the remaining portion of the sample was stored at -80°for verification.
Screening and characteristic analysis of lncRNA
In order to acquire the full transcriptome information from this sequencing, we used Cuffmerge software to merge the transcripts produced by splicing each sample and removed the transcripts with uncertain chain direction. The combined transcript collection was then screened for lncRNA, with the following major steps: 1) a large number of single exon transcripts with low expression and low confidence were selected in the splicing results of filtered transcripts, and those transcripts with exon number≥2 were selected. 2) the length≥200bp transcripts were selected. 3) Cuffcompare software was used to screen out transcripts that overlapped with the exon region of the annotated transcript in the database, and the lncRNAs that overlapped with the exon region of the spliced transcript in the database were included in the subsequent analysis as the annotated lncRNAs of the database. 4) the expression level of each transcript was calculated by Cuffquant, and the transcript with FPKM≥ 0.5 was selected. 5)screening transcripts with coding potential.
Differential expression and conservation analyses
Based on the concept of negative binomial distribution, EdgeR software was used to identify differential expression from digital transcripts or gene expression data. In the leg musculature of RC and CB, transcripts with a q adjusted value of 0.05 were found to be differentially expressed (DE). Two algorithms, FinFIT and phastCons (V1.3), were used in Phast (V1.3) to determine how much of a transcript's sequence is conserved15. Phylogenetic models of conserved and unconserved regions were calculated by phyloFit, and the running parameter was -- tree ‘(MM10, (galGal4, HG19))’. Then, the conserved fractions of a group of lncRNAs and coding genes with phastCons were calculated using the transformation parameters of the model and the hidden Markov model.
Target gene prediction and functional enrichment analyses
LncRNA target genes were predicted by correlation analysis or co-expression analysis of lncRNA and mRNA expression levels between samples. DE lncRNAs were selected for cis-target gene pre-dictions and were integrated with DE gene data to improve the veracity of target prediction. The study identified DE-mRNAs situated 10 kb upstream and downstream of DE-lncRNAs as cis-acting target genes and predicted their functional roles. All DE-mRNAs and target genes of DE-lncRNAs were annotated and classified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with OmicShare tools (HTTP://www. omics hare.com/tools/). The results with a p-value(P)<0.05 were considered significantly enriched. GO enrichment analyses of DE genes or lncRNA target genes were implemented with the GO seq-R package (Release 2.12)16. DE lncRNA genes were statistically enriched in KEGG pathways using the KOBAS (v2.0) software17. GO terms and KEGG pathways with corrected P<0.05 were considered significantly enriched in DE genes.
Rapid amplification of cDNA ends
The full-length sequence of lncRNA-004686 was obtained using a polymerase chain reaction (PCR) called Rapid Amplification of cDNA Ends (RACE). Following the manufacturer's instructions, a SMARTer RACE cDNA Amplification Kit (Takara, Tokyo, Japan) was used to amplify total RNA from breast muscle tissue for nested PCR. Sangon Biotech cloned (Shanghai, China) the RACE PCR results into the pUC19 vector (Takara, Tokyo, Japan) and sequenced them. The primers used for cloning the full-length chicken lncRNA-004686 are shown in Table 1.
Construction of overexpression vector and small interfering RNAs
The PCR products were then ligated into the pcDNA3.1(+) plasmid vector after being excised using the restriction endonucleases KpnI and XhoI. The pcDNA3.1(+)-004686 overexpression vector was given that name. Table 2 lists the siRNAs created and manufactured by Gene Pharma (Shanghai, China) for the targeted knockdown of lncRNA-004686.
Culture and transfection of chicken primary myoblasts
Following sterilization, leg muscle tissue was taken from E9. Primary myoblasts were identified and purified from breast muscle tissue using the differential adhesion technique, collagenase I, and trypsin digestion. Myoblasts were grown in high-glucose DMEM with 20% FBS (Gibco, Grand Island, USA) as an addition. Two days following cell confluence, myogenesis was induced using DMEM containing 2% HoS (HyClone, Logan, USA) and 1% penicillin-streptomycin. Following the manufacturer's recommendations, the transfection reactions were carried out using the Lipofectamine 2000 reagent (Invitrogen, California, USA) in Opti-MEM (Gibco, Grand Island, USA).
Dual-Luciferase Reporter Assay
HEK-293T cells were co-transfected with pcDNA3.1 (+) -004686 and the vectors of MYH1B. After 24 h of transfection, luciferase activity was detected using the Dual-Luciferase Reporter Assay Kit (Promega, Fitchburg, USA). The optical density of the resulting solution was assessed using an automatic microplate reader (Molecular Devices, Sunnyvale, USA).
Cell-Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EDU)assay
The cells were sown in a 96-well plate after being counted. When the cell density was between 50% and 60%, three replicates of each of the following were transfected into the cells: pcDNA3.1(+)-004686, pcDNA3.1(+), siRNA, siRNA-NC, and a blank control. Using a Cell Counting Kit-8, the cells' proliferation was observed after 24, 48, 72, and 96 hours (Biosharp, Anhui, China). Every 24 hours, CCK-8 solution was added to the medium, and after 1 hour of incubation, the OD at 450 nm was measured. At 48 h after transfection, the cells were incubated for 2h in 50μM EdU (RiboBio, Guangzhou, China). And then the cells were fixed in 4% paraformaldehyde for 30 min and stained with a C10310 EdU Apollo In Vitro Imaging Kit (RiboBio, Guangzhou, China). The ratio of the number of EdU-stained cells to the number of Hoechst 33342-stained cells was determined using images of three randomly selected fields obtained with a fluorescence microscope (TE2000-U; Nikon, Japan).
RNA Extraction and Real-Time qPCR
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was generated using the Prime Script TM RT reagent kit with gDNA Eraser (Takara, Tokyo, Japan). Real-time qualitative polymerase chain reaction (qPCR) for RNA studies were done using the SYBR Green PCR Master Mix (Takara, Tokyo, Japan).
Western Blot Analysis (WB)
RIPA Lysis Buffer (Solarbio, Beijing, China) containing protease inhibitors was used to extract proteins. Samples were denatured by heating, proteins were separated via SDS–PAGE Gel (EpiZyme, Shanghai, China) electrophoresis and transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany), sealed with 5% skimmed milk (BD, Franklin Lakes, NJ, USA) for 2 h in a shaker at room temperature, washed with TBST, and incubated overnight at 4 °C with Anti-myosin Skeletal Fast (Sigma, Darmstadt, GER), Anti-myosin Skeletal Slow (Sigma, Darmstadt, GER) and MYH (SANTA CRUZ, Santa Cruz, USA) antibodies. Next, the samples were washed thrice with TBST and incubated with the secondary antibody (Goat Anti-mouse(H+L) IgG, Boston, USA) at 37°C for 2 h. The protein bands on the membrane were quantified by the fluorescence system.
Statistical analysis
EXCEL software was used to collect and evaluate experimental data, while the 2-ΔΔCT method was used to calculate fluorescence quantitative PCR results. SPSS 22.0 software was used for one-way analysis of variance or independent sample T-test, Duancan's method for significance testing, and the Pearson method for correlation analysis. Results are presented as 'mean±standard deviation'.