Total RNA from tissue samples (frontal cortex, heart and testis) of one rhesus macaque animal and frontal cortex sample from one mouse animal was extracted using TRIzol reagent (Invitrogen, catalog #15596018) and analyzed on an Agilent 2100 Bioanalyzer to assess quality (Additional File 1 : Supplementary Table S4 ). The quantity of total RNA was measured by Qubit Invitrogen. cDNA was then synthesized using the SMARTer PCR cDNA Synthesis Kit (Clontech, catalog #634925) and PrimeSTAR GXL DNA Polymerase (Clontech, catalog #R050B). Single-molecule real-time (SMRT) bell libraries were generated by using an SMRTbell™ Template Prep Kit 1.0-SPv3 (PN 100-991-900) and subsequently sequenced by using Sequel Binding Kit 2.0, Sequel Sequencing Kit 2.1 and Sequel™ SMRT® Cell 1 M v2 Tray on the PacBio Sequel System (Pacific Biosciences). The Iso-Seq of cerebellum samples was performed as in our previous study [2 (link)] in which the sequencing was carried out on a real-time sequencer (Pacific Biosciences) with C4 sequencing reagents.
Protocol detail
Transcriptome Analysis of Rhesus Macaque Tissues
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TRIzol reagent
Agilent 2100 Bioanalyzer
SMARTer PCR cDNA Synthesis Kit
PrimeSTAR GXL DNA Polymerase
SMRTbell™ Template Prep Kit 1.0-SPv3
Sequel Binding Kit 2.0
Sequel Sequencing Kit 2.1
PacBio Sequel System
real-time sequencer
Animal
Cdna
Cell
Cerebellum
Dna polymerase
Frontal cortex
Heart
Mouse
Rhesus macaque
Synthesis
Testis
Tissue
Trizol
Corresponding Organization : Peking University
Other organizations : Fu Wai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College
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Variable analysis
independent variables
- Tissue samples (frontal cortex, heart and testis) of one rhesus macaque animal
- Frontal cortex sample from one mouse animal
- Total RNA quantity measured by Qubit Invitrogen
- Total RNA quality assessed on an Agilent 2100 Bioanalyzer
- TRIzol reagent (Invitrogen, catalog #15596018) used for total RNA extraction
- SMARTer PCR cDNA Synthesis Kit (Clontech, catalog #634925) and PrimeSTAR GXL DNA Polymerase (Clontech, catalog #R050B) used for cDNA synthesis
- SMRTbell™ Template Prep Kit 1.0-SPv3 (PN 100-991-900) used for single-molecule real-time (SMRT) bell library generation
- Sequel Binding Kit 2.0, Sequel Sequencing Kit 2.1 and Sequel™ SMRT® Cell 1 M v2 Tray on the PacBio Sequel System (Pacific Biosciences) used for sequencing
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