Animals collection and preparation
The experimental samples were collected from the Ningxia Yanchi Tan sheep breeding farm. Six unrelated Chinese Tan sheep (with no common grandparents) at two different physiological periods (the birth period and the Er-mao period) were selected and divided into the birth (BS) and Er-mao (ES) groups.
For each Tan sheep, after its hair was cut and the iodine was removed with alcohol, approximately 1 cm2 of skin tissue was taken with sterile forceps, and the tip of the sample was quickly cut with a sterile scalpel. After the procedures, Yunnan Baiyao (Yunnan Baiyao Group Co., Ltd., China) was used promptly to stop the Tan sheep bleeding. Half of each piece of Tan sheep skin tissue was stored in liquid nitrogen for RNA extraction, and the remaining half was stored in a 4% paraformaldehyde fixing solution for paraffin sectioning. All procedures including handing the Tan sheep samples were approved and implemented by the Laboratory Animal Care and Use Guide jointly developed by the Ministry of Agriculture of the People's Republic of China and the Institute of Animal Science of the Chinese Academy of Agricultural Sciences.
Skin tissue samples were cut into 1 cm*0.6 cm*0.6 cm pieces, washed under running water for 24 h, and dehydrated with different concentrations of alcohol. Then, the skin tissue was embedded in EPON 812 with an embedding machine and sliced continuously (5 microns) under an LKB 8800 ultratome to make paraffin sections, which were then stained with hematoxylin and eosin.
The skin tissues of three Tan sheep at both periods were selected for HE staining. Five different visual fields (100×) of each slice were randomly selected. The diameter of primary hair follicles and secondary hair follicles from the skin of Chinese Tan sheep in the birth and Er-mao periods were measured by ImageJ software. Specifically, the diameters of three primary hair follicles and five secondary hair follicles from sheep at each period were counted randomly, and the results were calculated as the mean ± standard deviation. Statistical analysis was carried out by R package, and one-way analysis of variance was used to analyze the differences in primary hair follicle and secondary hair follicle diameter at different time periods.
Rna Isolation And Sequencing
Six individual Tan sheep with complete skin tissue were selected from the ES (birth) and BS (Er-mao period) groups for whole-transcriptome sequencing (RNA-seq) analysis (N = 6 tissue samples). Total RNA was extracted from these 6 samples using the RNeasy Mini Kit (Qiagen, Germany) according to the reagent specification. Six skin samples with RNA integrity number (RIN) values above 6 were used for RNA-sEq. mRNA selection, library construction, and sequencing were performed using the Illumina HiSeq X10 platform at NovelBio Company (Shanghai, China).
We used TRIzol reagent (Thermo, USA) to isolate total RNA from skin tissues according to the manufacturer's instructions. The RNA quality and quantity were tested using a NanoDrop spectrophotometer.
Primary Processing And Mapping Of Rna-seq Reads
Raw reads were filtered using NGS QC Toolkit v2.3.3 software, and we removed reads with an adaptor sequence, reads whose ends were of low quality (Phred quality value < 20) and reads with a length less than 25 bp after filtration. The sheep reference genome Oar_v4.0 was downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/). The clean data were mapped to the sheep reference genome with TopHat v2.0.11 software .
Assembly Of Transcripts And De Analysis
Gene expression was quantified using Cufflinks (v2.2.1) software  to obtain FPKM expression values. We performed read alignment and quantitated expression for each sample. The output files were sent to Cuffmerge with the reference annotation files. We used the Cuffdiff package of Cufflink (v2.2.1) software to analyze the DEGs between Tan sheep in the birth and Er-mao periods. Genes with fold change > 1 or < -1 and a P value < 0.05 were identified as DEGs.
The FPKM expression values for all of the annotated transcripts from transcriptions of the skin of six Tan sheep were used to perform PCA, which was implemented by ggplot2 in R (R v3.6.1, https://www.r-project.org/).
Functional Enrichment Analyses
KEGG analysis  was a database using for understanding functions and utilities of the biological system. Pathways enriched in genes with an adjusted P value < 0.05 were considered significantly enriched pathways.
Mrna Expression Analysis
One microgram of RNA from each Tan sheep skin sample was reverse transcribed using the PrimeScript RT with gDNA Eraser kit (Takara, Japan) according to the operating instructions.
The primers used for qPCR, which were designed with the NCBI database (http://www.ncbi.nlm.nih.gov/), are listed in Table S1. qPCR was conducted in an ABI 7500 system (Applied Biosystems, USA) with a reaction volume of 20 µL that included 2 µL of cDNA template, 10 µL of 2 × SYBR Green Master Mix (RR420A, Takara), and 0.5 µL each of upstream and downstream primers. The PCR amplification procedure consisted of a 10 s cycle of 95 °C, followed by 40 cycles of 95 °C for 15 s and 62 °C for 34 s. The fluorescence of the products after each cycle was detected. qPCR for each gene consisted of three biological and technical replications, and the primer information is given in Table S1. ACTB was used as an internal reference gene. The 2−ΔΔCT  method was used to analyze relative mRNA expression.
Immunohistochemistry (the Sp Method)
Skin tissue sections were dehydrated and dewaxed with alcohol at different concentrations. After antigen repair, a 30 g/L H2O2 aqueous solution was added to block peroxidase for 10 min. The sections were incubated with normal goat serum albumin for 15 min, and each section was incubated with 50 µL of secondary antibody at 37 °C for 4 h. After washing with PBS, 50 µL of a biotin-labeled goat anti-rabbit IgG working solution was added to each section, and 50 µL of a horseradish enzyme-labeled streptomycin working solution was added. After the addition of DAB, the sections were dehydrated, cleared and sealed.
Five slices were randomly selected, and 5 different fields in each slice were selected (400×). Image-Pro Plus 6.0 software was used to calculate the average absorbance indicating the expression factor from the positive signal strength and positive area in different visual fields from immunohistochemical staining of the skin tissue of Chinese Tan sheep in the birth and Er-mao periods. Twenty-five data points were counted for each period, and the results are expressed as the mean ± standard deviation. Statistical analysis was carried out with the R package, and one-way analysis of variance was used to analyze differences in the expression level of the same factor at the two different time periods.