Ion Total RNA-Seq Kit v2 User Guide (Pub. no. 4476286 ... - URGV

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2TroubleshootingChapter 2 Prepare Whole Transcriptome LibrariesTroubleshootingObservation Possible Cause SolutionThe Agilent ® software does notcalculate one concentration and peaksizeLow yield and poor size distribution inthe amplified libraryLow yield in the amplified library andvery few differences in the Agilent ®2100 Bioanalyzer ® instrument tracesbefore and after you fragment the RNALow yield and no PCR productsThe software detects multiple peaks inthe amplified cDNA profileYou recovered
3Prepare Small RNA Libraries■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42■ Prepare the starting material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Guidelines for obtaining small RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Assess the amount and quality of small RNA in your total RNA samples . . . . . 43Enrich the samples for small RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Assess the quality and quantity of the small RNA‐enriched sample . . . . . . . . . . 48Determine the input amount . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48■ Construct the small RNA library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Hybridize and ligate the RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Perform reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Purify and size‐select the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52Amplify the cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54Purify and size‐select the amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56Assess the yield and size distribution of the amplified DNA . . . . . . . . . . . . . . . . 59■ Pool barcoded small RNA libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60■ Determine the library dilution required for template preparation. . . . . . . . . . . . 60■ Proceed to template preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61■ Typical size distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Plotted size distributions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Size distributions compared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64■ Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65Using a positive control to troubleshoot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65Ion Total RNA-Seq Kit v2 User Guide41
Page 1: USER GUIDEIon Total RNA-Seq Kit v2f
Page 6: Contents6 Ion Total RNA-Seq Kit v2
Page 11 and 12: Chapter 1 Product InformationKit co
Page 13 and 14: Chapter 1 Product InformationMateri
Page 15 and 16: 2Prepare Whole TranscriptomeLibrari
Page 17 and 18: Fragment the whole transcriptome RN
Page 19: Chapter 2 Prepare Whole Transcripto
Page 23 and 24: Chapter 2 Prepare Whole Transcripto
Page 29 and 30: e. For each sample, collect the elu
Page 33: Chapter 2 Prepare Whole Transcripto
Page 36 and 37: 2Chapter 2 Prepare Whole Transcript
Page 38 and 39: 2Chapter 2 Prepare Whole Transcript
Page 42 and 43: 3Chapter 3 Prepare Small RNA Librar
Page 68 and 69: AAppendix A Supplemental Informatio
Page 76 and 77: BAppendix B SafetyChemical safetyCh
Page 78 and 79: BAppendix B SafetyBiological hazard
Page 80: Documentation and SupportIon contac

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