Ion pi sequencing 200 kit v2

Total RNA of each sample was extracted with the Qiagen Rneasy Mini Kit and digested with Dnase I according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The integrity and size distribution of the RNA were verified with Agilent 2200 Bioanalyser (Agilent Technologies., USA.). Samples with the RNA Integrity Number ≥8.0 were used for cDNA library construction. The cDNA libraries for single-ending sequencing were constructed using Ion Total RNA-Seq Kit v2.0 (Life Technologies, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The cDNA libraries were then processed for the Proton Sequencing process according to the commercially available protocols. Samples were diluted and mixed, and the mixture was processed on a OneTouch 2 instrument (Life Technologies) and enriched on a OneTouch 2 ES station (Life Technologies) for preparing the template-positive Ion PI™ Ion Sphere™ Particles (Life Technologies) according to Ion PI™ Template OT2 200 Kit v2.0 (Life Technologies). After enrichment, the mixed template-positive Ion PI™ Ion Sphere™ Particles of samples was loaded on to 1 P1v2 Proton Chip (Life Technologies) and sequenced on Proton Sequencers according to Ion PI Sequencing 200 Kit v2.0 (Life Technologies).

The complementary DNA (cDNA) libraries for single-end sequencing were prepared using Ion Total RNA-Seq Kit v2.0 (Life Technologies) according to the manufacturer's instructions. After enrichment, the mixed template-positive Ion PITM Ion SphereTM Particles of samples was loaded on to 1 P1v2 Proton Chip (Life Technologies) and sequenced on Proton Sequencers according to Ion PI Sequencing 200 Kit v2.0 (Life Technologies) by NovelBio Corp. Laboratory, Shanghai.

Hepatoma cells were infected with AdGFP or AdPCK1 for 36 h and then RNA was extracted using TRIzol reagent (Invitrogen, Rockville, MD, USA) according to manufacturer’s instructions. RNA-seq and bioinformatic data analysis were conducted at Shanghai Novel Bio Ltd. (Shanghai, China). Strand-specific RNA-seq libraries were prepared using the Total RNA-seq (H/M/R) Library Prep Kit (Vazyme Biotech, Nanjing, China) and were sequenced on an Ion Torrent Proton Sequencer (Life Technologies, Carlsbad, CA, USA) according to the Ion PI Sequencing 200 Kit v2.0 (Life Technologies). Raw reads in FASTQ format were quality-controlled using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). RNA-seq reads were aligned to the reference genome using Bowtie and uniquely mapped reads were used for further analysis. Gene expression levels were expressed as reads per kilobase per million reads (RPKM) and differences in gene expression were calculated with rSeq (http://www-personal.umich.edu/~jianghui/rseq/).

RNA-seq and bioinformatic data analysis were performed by Shanghai Novelbio Ltd. Total RNA was extracted from each sample from vaginal tissues of WT mice and Loxl1 knockout mice (n = 4 in each group) by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Bioanalyzer 2200 (Aligent) and kept at −80°C. The RNA with RIN (RNA integrity number) >8.0 is acceptable for cDNA library construction. The cDNA libraries for single-end sequencing were prepared using Ion Total RNA-Seq Kit v2.0 (Life Technologies) according to the manufacturer’s instructions. Then the prepared library was loaded on to 1 P1v2 Proton Chip (Life Technologies) and sequenced on Proton Sequencers according to Ion PI Sequencing 200 Kit v2.0 (Life Technologies). Before read mapping, clean reads were obtained from the raw reads by removing the adaptor sequences. The clean reads were then aligned (version: Mfa5.0) using the MapSplice program (v2.1.6). In alignment, preliminary experiments were performed to optimize the alignment parameters (-s 22 -p 15–ins 6–del 6–non-canonical) to provide the largest information on the AS events [24 (link)].

Total RNA from the A549 cells with AFAP1-AS1 knockdown and control A549 cells were isolated and quantified. The complementary DNA (cDNA) libraries for single-end sequencing were prepared using Ion Total RNA-Seq Kit v2.0 (Life Technologies) according to the manufacturer’s instructions. The cDNA libraries were then processed for the Proton Sequencing process according to the commercially available protocols. Samples were diluted and mixed, the mixture was processed on a OneTouch 2 instrument (Life Technologies) and enriched on a OneTouch 2 ES station (Life Technologies) for preparing the template-positive Ion PI™ Ion Sphere™ Particles (Life Technologies) according to Ion PI™ Template OT2 200 Kit v2.0 (Life Technologies). After enrichment, the mixed template-positive Ion PI™ Ion Sphere™ Particles of samples was loaded on to 1 P1v2 Proton Chip (Life Technologies) and sequenced on Proton Sequencers according to Ion PI Sequencing 200 Kit v2.0 (Life Technologies) by NovelBio Corp. Laboratory, Shanghai. Data are available in Additional file 2: Table S2.