Agilent bioanalyzer 2100 system

Genomic DNA was isolated from the dorsal skin of mice from each of the four groups by using Qiagen DNeasy Blood and Tissue Kit (Qiagen). The quality of the isolated DNA was examined by using an Agilent 2100 Bioanalyzer System according to the manufacturer's protocols (Agilent Technologies). DNA that passed quality control was sheared into fragments ranging from 150 to 200 bp. Ends were repaired by using blunt‐ended fragments with 5′‐phosphorylated ends. Specific adaptors obtained from Illumina were ligated to the fragments by using adaptor‐modified ends. The unligated adapters were removed by using AMPure XP beads, and the purified samples were amplified by using PCR with SureSelect Primer and SureSelect Pre‐capture Reverse PCR primers to generate the library for further analysis. After hybridization was performed by using the exome capture library, samples were amplified with indexed tagging primers by using PCR. The quality and quantity of the enriched library were examined by using an Agilent 2100 Bioanalyzer System and real‐time PCR. The samples were then sequenced by using an Illumina Hiseq 4000 (Illumina) according to the manufacturer's standard protocols. A total of five gigabases of DNA base‐pair sequences were read, and the coverage of the whole‐exome sequencing was approximately 100‐fold.

For RNA isolation, 3 ml of whole blood were collected in Tempus™ Blood RNA Tubes (Thermo Fisher Scientific, USA) and further processed using Tempus™ Spin RNA Isolation Kit (Thermo Fisher Scientific, USA) according to manufacturer’s instructions. The quantity and quality of extracted RNA and prepared libraries were determined by Qubit Fluorometer (Thermo Fisher Scientific, USA) and Agilent 2100 Bioanalyzer systems (Agilent, USA), respectively. The integrity of RNA was evaluated by RNA integrity number (RIN) within the Agilent 2100 Bioanalyzer system (Agilent, USA). For depletion of ribosomal RNA 500 ng of total RNA from each sample were processed using Low Input RiboMinus™ Eukaryote System v2 (Thermo Fisher Scientific, USA). Complementary DNA library preparation was performed with Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific, USA). Ion Proton™ System (Post-Light™ Ion Semiconductor Sequencing, Thermo Fisher Scientific, USA) and Ion PI™ Chip (Thermo Fisher Scientific, USA) was used for 200-base-read single-end sequencing, following the manufacturer’s instructions. Since the shot-gun RNA-Seq is considered to be the most accurate and desirable method for the quantification of the individual transcript and gene expression, additional methods for technical validation were not applied in this study [30 ].

The leaf tissue of five-weeks-old seedlings of each genotypes was snap-frozen in liquid nitrogen and then kept at −80°C until RNA was extracted. A total RNA sample was extracted from ca100 mg leaf tissue of each genotype using the RNeasy Plant Mini Kit (#74904, QIAGEN, Valencia, CA), and subsequently treated with DNase using the Ambion Turbo DNA-Free Kit (#AM1907, Thermo Fisher Scientific, United States). An Agilent Bioanalyzer 2100 system (Agilent Technologies, United States), a NanoDrop ND-1000 spectrophotometer (Saveen Werner, Sweden), and agarose gel electrophoresis were used to evaluate the quality and concentration of extracted RNA. The high-quality RNA samples were then shipped to CD Genomics (New York, USA) on dry ice for RNA sequencing. Upon arrival at the CD Genomics, the samples were checked for degradation and contamination using agarose gels (1%), purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA), concentration was measured using the Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA), and integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The workflow for RNASeq data acquisation and analysis is provided in Figure 1.

Eggs, mixed larvae from 1st instar to before pupa stage, and mixed adults from emergence to egg laying period (10 days old) were prepared for RNA extraction. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). And RNA integrity was assessed using the RNA Nano6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq2000 platform and 100 paired-end reads were generated.

Total RNA was extracted from each pooled sample by using TRIzol (Ambion, United States) following the manufacturer’s instructions. Total RNA quantity and integrity were assessed by 1% agarose gel electrophoresis and the RNA Nano 6000 assay kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, United States).
A total amount of 2 μg high quality RNA per pooled sample was used for RNA sample preparation. All high-quality RNA samples were sent to BGI Co., Ltd. (Beijing, China). Sequencing libraries were constructed using the NEBNext^® Ultra^TM RNA Library Prep Kit for Illumina^® (NEB, United States) according to the manufacturer’s recommendations, and index codes were added to sequences to distinguish one sample from another. The Agilent Bioanalyzer 2100 system was employed to assess the quality of each RNA library. The index-coded samples were clustered 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 Hiseq platform and paired-end reads (Hiseq 4000, 101PE).

Extraction of total RNA from 6 cartilage tissues using the classical TRIzol extraction method, and the construction of 6 RNA libraries, were performed. The concentration and integrity of the total RNA were measured using a NanoPhotometer^® spectrophotometer (Thermo Scientific, Shanghai, China) and RNANano6000 Assay Kit from the Agilent Bioanalyzer 2100 system (Agilent, Beijing, China). Qualified RNA samples were used to construct RNA-seq libraries; the rRNA of those samples was removed using the NEB Next^® Ultra™ Directional RNA Library Prep Kit for Illumina ^® (NEB, Ipswich, MA, USA). PCR enriched cDNA were of approximately 150-200 bp length. Sequencing library quality was assessed using the Agilent Bioanalyzer 2100 system, and libraries meeting the quality criteria were sequenced using an Illumina Hiseq 4000 platform from Novegene Bioinformatics Technology Co., Ltd. (Beijing, China). The Illumina sequencing generated paired-end reads of 150 bp length.