ena-STUDY-Oxford Nanopore Technologies-30-08-2018-15:13:40:105-453
Oxford Nanopore RNA standards of differing known poly(A) tail lengths (10X-100X poly(A))
The aim of the study was to generate data from synthetic RNA samples with approximately known poly(A) tail lengths using the Oxford Nanopore MinION.RNA standard transcripts of differing known poly(A) tail lengths (10X-100X poly(A))DNA templates for in vitro RNA transcriptionLinear DNA templates for RNA transcription were made through a nested PCR protocol.  First, the Saccharomyces cerevisiae ORF for Enolase 2 (YHR174W) was isolated from genomic yeast DNA via PCR using specific primers (forward: 5'-GCCATCAGATTGTGTTTGTTAGTCGCTATGGCTGTCTCTAAAGTTTACG-3' and reverse: 5'-GCTTACGGTTCACTACTCACGACGATGTTACAACTTGTCACCGTGGTGG-3') (Integrated DNA Technologies, Coralville, IA, USA).  PCR was done in a final volume of 100 µl, with 1.2 ng/µl genomic DNA, 0.2 µM forward and reverse primers and 1X KAPA™ HiFi Hotstart DNA polymerase.  PCR conditions were 3 minutes at 95?C for initial denaturation, then 18 cycles as follows: 15 sec at 95?C, 15 sec at 62?C and 2 min at 72?C. The PCR amplicon was purified using 1X volume of Agencourt RNA Clean XP (Beckman Coulter), washed 2X with 200 µl of 70% EtOH, eluted in 33 µl of nH2O and quantified for a second round of PCR using a NanoDrop™ 2000c (Thermo Fisher Scientific) and Qubit™ 4 Fluorometer (dsDNA HS kit; Thermo Fisher Scientific). The second round of PCR was done with nested primers (forward: 5'-TAATACGACTCACTATAGGGAGAGCCATCAGATTGTGTTTGTTAGTCGCT-3' and reverse: 5'-[10-100X]T…GCTTACGGTTCACTACTCACGACGATG-3'), wherein the forward primer encodes a T7 RNA polymerase promoter site at the 5' end of the oligo and the reverse primers have either 10X, 15X, 30X, 60X, 80X, or 100X thymidine, on the 5' end.   An additional reverse primer was used (5'-[60X]T…[10X]N…GCTTACGGTTCACTACTCACGACGATG-3'), to generate DNA templates with 10 random nucleotides upstream of the 60X poly(A/T) region.  PCR conditions, amplicon purification and quantification were done the same as the first round PCR.  Additionally, the PCR amplicons were further assessed on a Bioanalyzer 2100 (12000 DNA kit; Agilent) to determine if there was a single species of homogenous product before proceeding with RNA transcription; if there was a smear of products or incorrect fragment size the sample would be rejected.  All reactions produced a single PCR amplicon of the correct size (1407-1417 bp).   In vitro RNA transcriptionRNA transcripts with 10X-100X poly(A) tails were generated individually using the PCR amplicon transcription templates for in vitro transcription and the MEGAScript™ T7 Transcription kit (Thermo Fisher Scientific), with 500 ng of starting DNA material in a 20 µl volume and a 4-hour incubation time at 37?C (as per kit instructions).  The DNA template was removed by adding 1 µl of TURBO DNase (supplied in kit) and incubating for 15 minutes at 37?C.  RNA transcripts were column-purified using the MEGAClear™ Transcription Clean-up kit (Thermo Fisher Scientific) as per kit instructions and eluted into a final volume of 100 µl.  The RNA transcription products were assessed using a NanoDrop™ 2000c and Qubit™ 4 Flourometer (HS RNA kit; Thermo Fisher Scientific).  Additionally, the transcripts were further assessed using a Bioanalyzer 2100 (6000 RNA Nano kit; Agilent) to determine if there was a single band of homogeneous product or a smear, indicating a distribution in the number of possible adenosines or a degraded sample.  If there was a smear of products or incorrect transcript size the sample would be rejected.  All transcription reactions produced a single polyadenylated RNA transcript of the correct size (1384-1474 nt).  Direct RNA sequencing The RNA transcripts with differing poly(A) lengths were sequenced individually using the SQK-RNA001 Direct RNA sequencing kit (Oxford Nanopore Technologies).  All sequencing sample preps were done with 500 ng of input material and the optional single-stranded (reverse transcription-free) protocol completed as per kit protocol instructions.  The samples were loaded onto R9.4.1 flowcells and sequenced on a MinION MK2 for 6 or 8 hours (MinKNOW v.1.10.16/Albacore v.2.0 sequencing and raw basecalling).
The aim of the study was to generate data from synthetic RNA samples with approximately known poly(A) tail lengths using the Oxford Nanopore MinION.RNA standard transcripts of differing known poly(A) tail lengths (10X-100X poly(A))DNA templates for in vitro RNA transcriptionLinear DNA templates for RNA transcription were made through a nested PCR protocol.  First, the Saccharomyces cerevisiae ORF for Enolase 2 (YHR174W) was isolated from genomic yeast DNA via PCR using specific primers (forward: 5'-GCCATCAGATTGTGTTTGTTAGTCGCTATGGCTGTCTCTAAAGTTTACG-3' and reverse: 5'-GCTTACGGTTCACTACTCACGACGATGTTACAACTTGTCACCGTGGTGG-3') (Integrated DNA Technologies, Coralville, IA, USA).  PCR was done in a final volume of 100 µl, with 1.2 ng/µl genomic DNA, 0.2 µM forward and reverse primers and 1X KAPA™ HiFi Hotstart DNA polymerase.  PCR conditions were 3 minutes at 95?C for initial denaturation, then 18 cycles as follows: 15 sec at 95?C, 15 sec at 62?C and 2 min at 72?C. The PCR amplicon was purified using 1X volume of Agencourt RNA Clean XP (Beckman Coulter), washed 2X with 200 µl of 70% EtOH, eluted in 33 µl of nH2O and quantified for a second round of PCR using a NanoDrop™ 2000c (Thermo Fisher Scientific) and Qubit™ 4 Fluorometer (dsDNA HS kit; Thermo Fisher Scientific). The second round of PCR was done with nested primers (forward: 5'-TAATACGACTCACTATAGGGAGAGCCATCAGATTGTGTTTGTTAGTCGCT-3' and reverse: 5'-[10-100X]T…GCTTACGGTTCACTACTCACGACGATG-3'), wherein the forward primer encodes a T7 RNA polymerase promoter site at the 5' end of the oligo and the reverse primers have either 10X, 15X, 30X, 60X, 80X, or 100X thymidine, on the 5' end.   An additional reverse primer was used (5'-[60X]T…[10X]N…GCTTACGGTTCACTACTCACGACGATG-3'), to generate DNA templates with 10 random nucleotides upstream of the 60X poly(A/T) region.  PCR conditions, amplicon purification and quantification were done the same as the first round PCR.  Additionally, the PCR amplicons were further assessed on a Bioanalyzer 2100 (12000 DNA kit; Agilent) to determine if there was a single species of homogenous product before proceeding with RNA transcription; if there was a smear of products or incorrect fragment size the sample would be rejected.  All reactions produced a single PCR amplicon of the correct size (1407-1417 bp).   In vitro RNA transcriptionRNA transcripts with 10X-100X poly(A) tails were generated individually using the PCR amplicon transcription templates for in vitro transcription and the MEGAScript™ T7 Transcription kit (Thermo Fisher Scientific), with 500 ng of starting DNA material in a 20 µl volume and a 4-hour incubation time at 37?C (as per kit instructions).  The DNA template was removed by adding 1 µl of TURBO DNase (supplied in kit) and incubating for 15 minutes at 37?C.  RNA transcripts were column-purified using the MEGAClear™ Transcription Clean-up kit (Thermo Fisher Scientific) as per kit instructions and eluted into a final volume of 100 µl.  The RNA transcription products were assessed using a NanoDrop™ 2000c and Qubit™ 4 Flourometer (HS RNA kit; Thermo Fisher Scientific).  Additionally, the transcripts were further assessed using a Bioanalyzer 2100 (6000 RNA Nano kit; Agilent) to determine if there was a single band of homogeneous product or a smear, indicating a distribution in the number of possible adenosines or a degraded sample.  If there was a smear of products or incorrect transcript size the sample would be rejected.  All transcription reactions produced a single polyadenylated RNA transcript of the correct size (1384-1474 nt).  Direct RNA sequencing The RNA transcripts with differing poly(A) lengths were sequenced individually using the SQK-RNA001 Direct RNA sequencing kit (Oxford Nanopore Technologies).  All sequencing sample preps were done with 500 ng of input material and the optional single-stranded (reverse transcription-free) protocol completed as per kit protocol instructions.  The samples were loaded onto R9.4.1 flowcells and sequenced on a MinION MK2 for 6 or 8 hours (MinKNOW v.1.10.16/Albacore v.2.0 sequencing and raw basecalling).