Agilent 2100

All plants of pineapple cultivar “Shenwan” used for this study were cultivated in Shenwan Town, Zhongshan City, Guangdong, China. Three distinct plant stages were defined according to fruit development. Stage 1 begins when flower buds begin to bloom and the collective fruit begins to expand in size. Stage 2 begins when flowering ceases, and the collective fruit expands at its fastest rate. Stage 3 corresponds to the time when collective fruit stops expanding and begins to ripen. In total, 11 tissues were manually separated from pineapple. These included bract, core, flower disk, leaf, ovary wall, ovule, petal, placenta, receptacle, sepal, stamen, and style tissues. All samples were collected and stored immediately in liquid nitrogen before RNA extraction. We extracted total RNA from all samples using TRIzol (Invitrogen, Carlsbad, CA, USA) and determined RNA quality of samples using Agilent 2100. RNA samples with RIN numbers lower than eight were re-extracted. All RNA samples were then forwarded to a sequencing provider for RNA sequencing.

Total RNA was extracted from the leaf samples. A Nanodrop 2000 (Thermo Fisher Scientific, Austin, TX, USA) was used to determine the concentration and purity of the extracted RNA. Subsequently, 1% agarose gel electrophoresis was conducted to evaluate the genomic contamination, purity, and RNA integrity of the samples. An Agilent 2100 (Agilent Technologies, Palo Alto, CA, USA) was used to determine the RNA integrity number (RIN) value. The full-length cDNA of the extracted mRNA was synthesized using a Clontech SMARTer PCR cDNA Synthesis Kit (Clontech Laboratories, Frederick, MD, USA). Primers with Oligo dT were used to pair with the A-T bases at the polyA region for the reverse transcription of the mRNA to obtain cDNA. The full-length cDNA was amplified by PCR, and the product was purified using PB magnetic beads to remove fragments of cDNA that were less than 1 kb. The end of cDNA was repaired, and the fragments were connected with the SMRT dumbbell connector. The unconnected fragments were digested using exonuclease and purified using PB magnetic beads to obtain the sequencing library. A Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA, USA) was used to accurately quantify the library, and an Agilent 2100 was used to determine the library size. The DNA was sequenced after the library size met the criteria.

Three experimental replicates from 9311 (ACK, AT1, AT1H1, AT3, and AT3H1) and DC907 (BCK, BT1, BT1H1, BT1H3, BT3, BT3H1, and BT3H3) cultivars were used for sequencing. Total RNA was extracted using an RNA Prep Pure Plant Kit (TIANGEN, Beijing, China) following the manufacturer’s protocol. RNA integrity was detected with an Agilent 2100 (Agilent Technologies, CA, USA). Oligo-dT primers and Array Script reverse transcriptase were used to construct the cDNA library. After library construction, initial quantification was performed using Qubit 2.0 to dilute the library to 1 ng/µL (Life Technologies, CA, USA), and then the insert size of the library was detected with Agilent 2100. After the insert size met expectations, the effective concentration of the library was quantified accurately by q-PCR (library effective concentration > 2 nM) to ensure library quality. All samples produced 150 paired terminal-sequence readings on the Illumina platform (Hiseq-PE150).

RNA with Poly-A structure in eukaryotic total RNA was enriched using the TIANSeq mRNA capture kit (TIANGEN Biotech). Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) was used to determine the concentration of RNA samples and assess their purity^{61 (link)}. Agilent 2100 Bioanalyser and 2100 RNA nano 6000 Assay Kit (Agilent Technologies) were used to assess the integrity of the RNA samples^{62 (link)}. Using the captured RNA as the starting sample, TIANSeq Fast RNA Library Kit (Illumina) was used to construct the transcriptome sequencing libraries. The transcriptome sequencing library was constructed through RNA randomly fragmentation, cDNA strand 1/strand 2 synthesis, end repair, A-tailing, ligation of sequencing adapters, size selection and library PCR enrichment. Library concentration was first quantified using Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/µl before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity > 2 nM). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina sequencing platform and 150 bp paired-end reads were generated.

All 30-days-old (sexually immature) and 120-days-old (sexually mature) HZ boars, and LC boars at the same age were castrated to obtain the testes samples. The testes on the same side were immediately frozen in liquid nitrogen and then stored at−80°C until further use. Total RNA was extracted from pooled samples by Trizol reagent kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer's instructions. Quantity and integrality of total RNA was detected by Agilent 2,100 (Agilent, United States) and NanoDrop 2,000 (Thermo Scientific, United States) instruments, and small RNA obtained from total RNA was used to construct small RNA libraries were then tested for quality and yield using Agilent 2,100 and ABI StepOnePlus Real-Time PCR System (Life Technologies). RNA with a RIN ≥7 was used to construct small RNA libraries and subsequent sequencing. The average RIN value of the RNA samples extracted from all testis tissues was 8.4 ± 0.70.

Total RNA was extracted using TRIzol reagent. The concentration and integrity of the RNA were measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). RNAs of 18–30 nt were enriched by polyacrylamide gel electrophoresis. Six libraries (three for the M group, three for the C group) were constructed by the NEBNext small RNA library prep set (New England Biolabs; E7300). Briefly, the 3’ and 5’ adapters were added to the RNAs. Then, the RNAs were reverse transcribed and amplified by PCR. The PCR products (140–160 bp in length) were recycled for sequencing. The libraries were evaluated by the Agilent 2100 and the ABI StepOnePlus Real-Time PCR System (Life Technologies, CA, United States). Finally, the libraries were sequenced by Illumina Hiseq2000 (Illumina, San Diego, CA, USA).