Smarter pcr cdna synthesis kit

The Iso-Seq library was prepared using the Isoform Sequencing protocol (Iso-Seq) with the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Pacific Biosciences (P/N100–377–100-05 and P/N100–377–100-04) with some modifications. First, 3 μl of total RNA solution was added to deionized water containing a single primer and incubated at 72 °C for 3 min (3′ SMART primer IIA from the Clontech SMARTer kit 5′–AAGCAGTGGTATCAACGCAGAGTACTNN–3′). Next, the SMARTER II A oligonucleotide (from the Clontech SMARTer kit 5′-AAGCAGTGGTATCAACGCAGAGTACXXXXX–3′), 5X first-strand buffer, DTT, dNTP mix, RNase inhibitor and SMARTScribe reverse transcriptase were added to the mixture and incubated at 72 °C for 1 h. Finally, the reaction was terminated at 70 °C. After 23 PCR cycles, the length of the PCR product was screened by the BluePippin Size Selection System and divided into 1–2 Kb, 2–3 Kb and 3–10 Kb fragments. After size screening, the cDNA was subjected to 12 cycles of PCR reactions. The PCR amplification products were used to construct the SMRTbell Template libraries using the Iso-Seq protocol. The libraries were prepared for sequencing by annealing a sequencing primer (component of the SMRTbell Template Prep Kit 1.0) and binding polymerase to the primer-annealed template.

We constructed FL transcriptome sequencing library of 1–6 kb complementary (cDNA) for the mixed pool sample. The library was sequenced on one SMRT Cell of Pacific Biosciences (PacBio) platform. Briefly, SMARTer™ PCR cDNA Synthesis Kit (Pacific Biosciences, Menlo Park, CA, USA) was used to generate first- and second-strand cDNA from mRNA. After a round of polymerase chain reaction (PCR) amplification and end repair, SMRTbell™ hairpin adapters were ligated. By exonuclease digestion, we obtained a 1–6 kb cDNA library.
Twelve libraries of two tissues and two sexes (three duplicates) were constructed following the protocol of the Gene Expression Sample Prep Kit (Illumina, San Diego, CA, USA). The libraries were sequenced by Illumina NovaSeq S4 platform with paired-end (PE) 150 nt.

Stool was collected from TA-treated or untreated mice and extracted using PowerFecal DNA isolation kit (MOBIO). The Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Pacific Biosciences (PN 100-092-800-03). Sequencing was performed to obtain DNA (125  bp) paired end reads to a depth of 10G base pairs per sample using HiSeq Illuminex 2500. Readfq (V8, https://github.com/cjfields/readfq) was used for preprocessing raw data from the Illumina sequencing platform to obtain clean data for subsequent analysis differential expression analysis of two groups was performed using the DESeq R package (1.18.0). DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting p-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted p-value < 0.05 found by DESeq were assigned as differentially expressed. LEfSe software is used for LEfSe analysis based on the the abundance at each taxonomy level.

Total RNA from each sample was extracted using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa Bio Inc., Kusatsu, Shiga, Japan). The RNA integrity value (RIN) was checked with a Bioanalyzer 2100 (Agilent Technologies Co., Ltd., Palo Alto, CA, USA). The total RNA quality was assessed by means of 1.2% agarose gel electrophoresis. The RNA samples with RIN > 8 were used for subsequent analyses.
For the Iso-Seq analysis, RNA from the Y and R samples were extracted separately and then mixed equally into one sample (designated R1). Similarly, the W and S extracts were mixed to form the S1 sample. The Iso-Seq library was prepared in accordance with the isoform sequencing protocol using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol, as prescribed by Pacific Biosciences (PN 100-092-800-03), on a PacBio platform by Novogene Co., Ltd. (Beijing, China).
Total RNA from the taproots of the four materials was extracted for each sample and used as the input material to create sequencing libraries using the NEBNext Ultra^TM RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) [58 ], following the manufacturer’s instructions, and deep sequenced by Novogene Co., Ltd. (Beijing, China) using an Illumina sequencing platform.

Embryos were observed under microscopes, and developmental stages were determined according to the developmental stages of common goldfish [3 (link),7 (link)]. Embryos at three stages, i.e., zygote period, 14-Somite stage, and 35% optic vesicle closure (OVC) stage were collected and subjected directly to RNA preparation using Trizol reagent. Five embryos were used for each stage. Total RNA quality was determined by electrophoresis. RNA from the three developmental stages were mixed equivalently, before library construction and sequencing on the PacBio platform. The Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol, as described by Pacific Biosciences (PN 100-092-800-03). For gene expression analysis, RNA from each stage of Ryukin and Celestial-Eye goldfish were subjected to library preparation and sequencing separately, according to the protocol. RNA-seq was performed on the HiSeq 2000 platform.