Smarter polymerase chain reaction cdna synthesis kit

The Iso-Seq method was used for sequencing long-reads of even proportions of tissue from gonads. The quality of extracted RNAs was evaluated using an Agilent 2100 Bioanalyzer (SA Pathology, Adelaide, SA, Australia). High-quality RNAs (RNA integrity number >6.0) were used for cDNA synthesis. The Iso-Seq library was prepared according to the Iso-Seq protocol using Oligo (dT) enriched mRNA. A Clontech SMARTer polymerase chain reaction (PCR) cDNA Synthesis Kit was used to reverse-transcribe mRNA into DNA. The BluePippin system was used to perform fragment screening of the full-length cDNA for damage repair, end repair, the connection of SMRT dumbbell joints, and exonuclease digestion. The Iso-Seq library was prepared as described by Pacific Biosciences (PN 100-092-800-03). Single-Molecule Real-Time (SMRT) sequencing was performed on the PacBio Sequel System.

Total ribonucleic acid (RNA) was extracted (RNA Easy Plus Kit; Qiagen, Hilden, Germany) and quantitated (Qubit^® 2.0 fluorometer; Life Technologies, Milan, Italy) for suppression subtractive hybridization (SSH). In two patient groups (ATBD, LTBI), 300 ng total RNA from each individual were pooled and quantified. Pooled total RNA (300 ng) was used for complementary deoxyribonucleic acid (cDNA) synthesis (SMARTer^™ polymerase chain reaction [PCR] cDNA Synthesis Kit; Clontech Laboratories, Inc., Palo Alto, California, United States).

In order to obtain the complete information of full-length transcriptome, total RNA from 19 different samples were pooled together in an equal quantity to construct libraries for PacBio sequencing. The mixed RNA sample was reverse transcribed using the Clontech SMARTer polymerase chain reaction (PCR) cDNA Synthesis Kit and oligo (dT). Large-scale PCR amplification was carried out to generate barcode full-length cDNA. The BluePippin Size Selection System protocol as described by Pacific Biosciences (PN 100-092-800-03) was used to separate the size of PCR selection for mix sample >4 kb. After damage repairing and end joining, the full-length cDNA is ligated to the SMRT dump bell joint. These cDNA products were purified for Iso-Seq SMRTBell library preparation. A SMRT cells were sequenced on the PacBio Sequel System platform.