Ethics approval and consent to participate
All animal experiments were conducted according to the guidelines established by the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, 2004). The tissues were collected from 43-week-old hens. The hens were raised under free food intake and were humanely sacrificed in the laboratory.
Three eggs from 1820 hens (Jianghan chicken, which is 43-week-old and is a local chicken in China) were collected. Eggshell breaking strength, eggshell thickness and egg weight were assessed. The eggshell thicknesses of eggs were tested using an electronic digital micrometer (Deli, Deli Group Co., Ltd, China). Eggshell breaking strength was assessed using an eggshell strength meter (NFN388, FHK Fujipin Co., Ltd, Japan). Eggs were weighed using an electronic balance (ES-E, Tianjin de ante Sensing Technology Co., Ltd, Tianjin, China).
The uteri of hens producing eggs with extreme eggshell thickness and eggshell breaking strength were collected immediately after oviposition from six hens after surgery. There were three uterus samples from the hard-eggshell group (HE group, exhibiting strong and thick eggshells) and three uterus samples from the week-eggshell group (LE group, exhibiting weak and thin eggshell). Then, the uteri were frozen in liquid nitrogen and used for RNA-seq analysis and experimental validation.
Total RNA was isolated with TRIzol reagent (Invitrogen, USA). After total RNA was isolated from each individual sample and assessed, the concentration, quality and integrity were determined by a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). A total of 5 µg RNA from each sample was employed for sequencing and library construction, which were performed at Novogene Biotech Co., Ltd. (Beijing, China).
The cDNA libraries were constructed using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) according to the instructions of the manufacturer. Poly(A) mRNA was isolated from purified total RNA using biotin-oligo (dT) magnetic beads and fragmented to generate average insert sizes of approximately 350 bp before the cDNA libraries were created. Quality control steps were undertaken using Qubit2.0 fluorescence spectrophotometry and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). A cluster was generated, diluted to 1.5 ng/µL, and sequenced using the Illumina PE150 platform (Illumina, San Diego, CA) with paired-end 2 × 150-bp reads. Sequences were mapped to the chicken reference genome database from the NCBI database (ftp.ncbi.nlm.nih.gov/genomes/genbank/vertebrate_other/Gallus_gallus/latest_assembly_versions/GCA_000002315.5_GRCg6a/) with TopHat2 . All RNA-seq data were uploaded to the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/PRJNA660090; SRA accession number: PRJNA660090).
Protein extraction, TMT labeling and LC MS/MS analysis
Total proteins from the same sample of RNA-seq were extracted by the cold acetone method. The supernatant from each sample, containing precisely 0.1 mg of protein, was digested with Trypsin Gold (Promega, Madison, USA) at a 1:50 enzyme-to-substrate ratio. After 16 h of digestion at 37 °C, peptides were desalted with a C18 cartridge to remove the high urea, and desalted peptides were dried by vacuum centrifugation.
Desalted peptides were labeled with TMT6/10-plex reagents (TMT6/10plex™ Isobaric Label Reagent Set, Thermo Fisher) following the manufacturer’s instructions. Differently labeled peptides were mixed equally and subsequently desalted by peptide desalting spin columns (Thermo Fisher, 89852). TMT-labeled peptide mix was fractionated using a C18 column (Waters BEH C18 4.6 × 250 mm, 5 µm) on a Rigol L3000 HPLC operating at 1 mL/min, and the column oven was set at 50 °C. Mobile phases A (2% acetonitrile, adjusted pH to 10.0 using ammonium hydroxide) and B (98% acetonitrile, adjusted pH to 10.0 using ammonium hydroxide) were employed to develop a gradient elution. The eluates were monitored at UV 214 nm, collected into a tube each minute and merged into 10 fractions. All fractions were dried under vacuum and reconstituted in 0.1% (v/v) formic acid (FA) for subsequent analyses.
Shotgun proteomics analyses were performed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) operated in the data-dependent acquisition (DDA) mode.
Proteins with fold change in a comparison ≥ 1.2 or ≤ 0.83 and unadjusted significance level p < 0.05 were considered to be differentially expressed. Gene Ontology (GO) and InterPro (IPR) analyses were conducted using the interproscan-5 program against the nonredundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSiteProfiles, and PANTHER) , and the KEGG database (Kyoto Encyclopedia of Genes and Genomes) was employed to analyze the protein family and pathway. The probable interacting partners were predicted using the STRING-db server (http://string-db.org/) based on the related species. STRING is a database of both known and predicted protein-protein interactions . The enrichment pipeline  was utilized to perform the enrichment analysis of GO and KEGG.
Correlation analysis between proteomic and transcriptomic results
The differentially expressed genes (DEGs) and the differentially expressed proteins (DEPs) were identified separately, and Venn diagrams were plotted according to the counted results. Correlation analysis was performed by R (version 3.5.1), and the maps were drawn based on changes in the transcriptome and proteome analyses.
Quantitative real-time PCR analysis of differentially expressed genes
RNA samples from the three chickens used for the RNA-seq experiment were analyzed by qRT-PCR. The concentrations of RNA samples were measured using a NanoQuant Plate (TECAN, Infinite M200PRO). Total cDNA was synthesized using a PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China). qRT-PCR was performed with a LightCycle® 480 II (Roche) in a final volume of 20 µL using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). Each 20-µL reaction volume included 10 µL of THUNDERBIRD SYBR qPCR Mix, 0.3 µL of each divergent primer, 1 µL of cDNA and 8.4 µL of double-distilled H2O (ddH2O). The cycling conditions included an initial single cycle (98 °C for 10 s) followed by 40 cycles (94 °C for 15 s and 60 °C for 30 s). β-actin was used as an internal control for the expression of mRNA. The relative expression level was normalized and calculated using the 2−ΔΔCt method . All primers were designed using Primer 5 (listed in Supplementary Table S1).
Western blot analysis
RIPA lysis buffer (Beyotime, Beijing, China) was employed to generate uterine protein lysates. The uterine protein was extracted and separated using SDS-PAGE and transferred to PVDF membranes, which were blocked with skim milk. Then, antibodies specific for differential proteins (1:2000-1:1000, Abcam, Cambridge, MA, USA) and β-actin (1:1000, Abcam, Cambridge, MA, USA) were used for immunoblotting. We quantified the protein expression levels compared to β-actin expression using ImageJ 1.42q (Wayne Rasband, National Institutes of Health, USA).
All data analyses were performed by GraphPad Prism 5 (San Diego, CA). The experimental values are presented as the means ± SDs. The means of the groups were compared by Student's t-test. P-values < 0.05 were considered to indicate significance.