Thyroid transcriptomic profiling reveals the differential regulation of lncRNA and mRNA related to prolificacy in Small Tail Han sheep with FecB⁺⁺ genotype

Figures & data

Table 1. Reverse transcription using reagents and the volume used.

Reagents	Volume (µL)
PrimeScript RT Enzyme Mix I	1
Oligo dT Primer	1
Random 6 Mers	1
5× PrimeScript Buffer	4
Total RNA (500 ng/µL)	2
RNase-free ddH2O	11

Table 2. Primer sequences.

Gene name	Classification	Sequence (5′–3′)
LNC_004319	F	ACCATCGTTTGCCCATCTCTCTTTC
	R	GATCACGGTCAAGGTCACCATCAAG
LNC_012932	F	GACAGCCAAGCGGACGACTATG
	R	AGCCTGAGGACTACAAGCCCAAG
LNC_005171	F	TCCAACACAACTGAGCGACTAAGC
	R	GGGTAAAGGGTCTGAGCCAACATTG
LNC_003561	F	TCTGGGTTTGGTGCTTGGTGTAAG
	R	CTCGTGGCTGCTTCCTCTTGTG
LNC_012999	F	TCAGGAAGGCAGCAAGCAGAAAC
	R	CAGGCGGTGATTCAGTATGGGAAG
CACNB3	F	AGCATGGCATGAGGGAGAAACAAG
	R	TGTGGCAGTGAGGTGAGTAGAGG
DMD	F	GCAAGAGCACAACAATCTGGTCAAC
	R	ATCCTCCCTGTTCGTCCCGTATC
ASCC1	F	AGTTCCTGTTCCTTCACCCTACCC
	R	AATTGCTGCTGCCCTGGTAAGTAG
SYNPO2	F	TTGTTGGGCAGCAGGGTTTGAG
	R	CAGACACATCCATCCACCACTTCC
REPS2	F	TGAGCCTCCCTACTTTGTTGTTTGC
	R	GCCCTCTTGGTCTGCTGTTCTTAG
RPL-19	F	ATCGCCAATGCCAACTC
	R	CCTTTCGCTTACCTATACC

Table 3. qPCR reagent usage.

Reagents	Volume (µL)
SYBR Premix Ex Taq II	10
Forward primer	0.8
Reverse primer	0.8
cDNA	2
RNase-free ddH2O	6.4

Table 4. Condition setting of qPCR.

Cycles	Temperature (°C)	Time
1×	95	5 min
40×	95	10 s
	60	10 s
	72	10 s
1×	95	5 s
	65	1 min
1×	40	30 s

Table 5. Summary of raw reads after quality control and Mapping to the reference genome.

Sample name	Raw reads	Clean reads	Clean bases	Mapped reads	Mapping rate (%)	Q30 (%)
ww_F_T_1	90,959,354	88,524,974	13.28G	81,088,654	91.6	94.29
ww_F_T_2	103,513,712	99,977,788	15G	90,093,436	90.11	92.7
ww_F_T_3	84,148,306	80,737,376	12.11G	72,225,658	89.46	92.46
ww_L_T_1	89,888,342	87,939,706	13.19G	80,503,075	91.54	92.89
ww_L_T_2	105,656,302	101,603,048	15.24G	91,764,845	90.32	92.98
ww_L_T_3	106,622,786	102,672,444	15.4G	91,299,933	88.92	91.15

Figure 1. (A) The distribution of lncRNAs and mRNAs in different chromosomes. (B) The length statistics of lncRNAs and mRNAs. (C) The statistics of lncRNAs and mRNAs exon number. (D) Classification of lncRNAs, including lincRNAs, intronic-lncRNAs, lncRNA and antisense_lncRNAs.

Figure 2. (A) Number of up-and down-regulated lncRNAs and mRNAs in the sheep thyroid between ww_FT and ww_LT. (B) Overlapping numbers of genes targeted by DELs and DEGs in ww_FT and ww_LT. 96 DELs (C) and 1054 DEGs (D) in ww_FT and ww_LT are shown in the heat map.

Figure 3. The top 30 enriched GO terms for the identified genes targeted by DELs (A) and DEGs (B) in ww_FT and ww_LT.

Figure 4. The 15 enriched KEGG pathways for the identified genes targeted by DELs (A) and DEGs (B) in ww_FT and ww_LT.

Figure 5. Co-expression of lncRNAs–mRNAs after genes targeted by DELs coincided with DEGs in ww_FT and ww_LT. Red (increased expression during the follicular phase) and green (decreased expression during the follicular phase) indicate up- or down-regulation, respectively.

Figure 6. RT-qPCR validation of DELs (A) and DEGs (B) identified by RNA-seq in ww_FT and ww_LT (P < .05).

Data availability statement

Experimental animals in this study were authorized by the Science Research Department (in charge of animal welfare issues) of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (IAS-CAAS; Beijing, China). Additionally, ethical approval of animal survival and the sample collection was given by the animal ethics committee of IAS-CAAS (No. IAS2019-49).