Poly t oligo attached magnetic beads

Total RNA was extracted from each sample using an RNAprep pure Plant Kit (Tiangen, Beijing, China), according to the manufacturer’s protocol. The RNA concentration of each sample was measured using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). The RNA quality was assessed using an Agilent2200 (Agilent Technologies, Santa Clara, CA, USA).
The sequencing library for each RNA sample was prepared using a TruseqTM RNA sample prep Kit (Illumina, San Diego, CA, USA), following the manufacturer’s protocol. Briefly, mRNA was purified using poly-T oligo-attached magnetic beads (Invitrogen,Carlsbad, CA, USA) from 5 μg total RNA. The mRNA was fragmented, and the RNA fragments were reverse transcribed and amplified to double-stranded cDNA. Index adapters were then ligated to the cDNA according to the protocol of the TruseqTM RNA sample prep Kit (Illumina). The library was quantified using a TBS-380 mini-fluorometer (Picogreen, Cohasset, MA, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System, using a 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 2500 platform and a sequence length of 2*101 bp paired-end reads were generated.

Total RNAs of goose follicles were extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s procedure. Their quantity and purity were analyzed by the Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RNA integrity >7.0. Approximately 5-μg total RNA of each sample was subjected to isolating Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were constructed into the cDNA library according to the protocol for the Illumina RNA ligation based method (Illumina, San Diego, USA). We performed the single end sequencing on an Illumina Hiseq 2500 at the LC Sciences, China, following the vendor’s recommended protocol.

Samples from LNM⁻ primary tumors (n = 4) and LNM⁺ primary tumors (n = 5) were cut into small specimens. The total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s procedure. The purity and quantity of total RNA were analyzed using NanoDrop ND 1000 (NanoDrop, Wilmington, DE, USA), and the integrity of the RNA was assessed using Agilent 2100 with RIN number >7.0. Poly(A) RNA was purified from total RNA (5 µg) using poly T oligo attached magnetic beads using two rounds of purification (Invitrogen). The mRNA was then fragmented into small pieces using divalent cations under elevated temperature. Subsequently, the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-sequencing sample preparation kit (Illumina, San Diego, CA, USA). Lastly, we performed the 150-bp paired-end sequencing on an Illumina X Ten (LC Bio, Hangzhou, China) following the recommended protocols.

Total RNA was extracted using the TRIzol Reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analyzed using the 2100 Bioanalyzer and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number > 7.0. Approximately 10 μg of total RNA representing a specific adipose type was subjected to isolate poly(A) mRNA with poly-T oligoattached magnetic beads (Invitrogen). Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then, the cleaved RNA fragments were reverse transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA); the average insert size for the paired-end libraries was 300 bp (±50 bp). And then we performed the paired-end sequencing on an Illumina Hiseq 4000 at LC Sciences, USA, following the vendor's recommended protocol.

We randomly picked three pigs in every group for deep sequence analysis. Trizol reagent was used for RNA isolation from blood vessels at the end of abdominal aorta (Invitrogen, CA, USA). RNA quantity and purity were assessed using a Bioanalyzer 2100 and RNA 6000 Nano Lab Chip Kit (Agilent, CA, USA) with RNA integrity number (RIN) > 7.0. Ten micrograms of total RNA was used to isolate Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). After purification, the mRNA was cleaved into small fragments by divalent cation at high temperatures. According to the protocol of the mRNA Seq sample preparation kit (Illunina, San Diego, USA), the RNA fragments after cleavage were reverse-transcribed to generate the final cDNA library. The average insertion size of the paired-end library was 300 bps. We then performed the paired-end sequencing on Illumina Hiseq 4000 (LC Sciences, Hangzhou, China) as recommended by the supplier. RNA-Seq data have been submitted to the Gene Expression Omnibus (GEO) database (Accession no.GSE140412).

Total RNA of U87-MG, U251-MG and normal brain was extracted using Trizol reagent (Invitrogen) following the manufacturer's procedures. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, Santa Clara, USA). Roughly 10 μg of total RNA was subjected to isolate Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). Following purification, the mRNA is fragmented into small pieces using divalent cations under raised temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina). The average insert size for the paired-end libraries was 300 bp (± 50 bp). Next we performed the paired-end sequencing on an Illumina Hiseq 2,000 system at Macrogen (Seoul, Korea) following the vendor's recommended protocol. For each sample, sequenced reads were aligned to the UCSC human reference genome (http://genome.ucsc.edu/) using the Tophat package (http://ccb.jhu.edu/software/tophat), which initially removes a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome. FPKM (fragments per kilo base of exon per million fragments mapped) were calculated to compare the expression level of AMACR mRNA variants in each sample.

Total RNA was extracted from the skin of fish in the control group and the 16-h transport group using Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The quantity and purity of RNA were checked using a Bioanalyzer 2100 and RNA 6000 Nano Lab Chip Kit (Agilent, Palo Alto, CA, USA) with RIN >7.0. The poly (A) mRNA was isolated from approximately 10 µg RNA with poly-T oligo-attached magnetic beads (Invitrogen). The mRNA was fragmented into small pieces using divalent cations. After purification, the cleaved RNA fragments were reverse-transcribed to build the final cDNA library according to the protocol of the mRNA Seq sample preparation kit (Illumina, San Diego, CA, USA). The average insert size was 300 bp ( ± 50 bp) for the cDNA libraries. Following the manufacturer’s recommended protocol, the transcriptomes were sequenced, generating paired-end reads of 150bp length on the IlluminaHiseq4000 platform at LC Sciences (Houston, TX, USA) using the Illumina paired-end RNA-seq approach.