Total RNA from the WTC11 passage 79 pellet was extracted using the RNeasy Kit (Qiagen). This extracted RNA was analyzed on an Agilent Bioanalyzer to confirm sufficient concentration and quality of RNA for downstream data generation. As described by our group previously,24 (link) complementary DNA (cDNA) was synthesized from the extracted RNA, and the Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 (Pacific Biosciences) was used on a Sequel II system to obtain long-read sequence information and output Circular Consensus (CCS) reads.
Iso seq express kit smrt bell express template prep kit 2
Manufactured by Pacific Biosciences
The Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 is a laboratory equipment product designed for sample preparation in long-read sequencing workflows. It provides a streamlined process for generating SMRT Bell libraries from RNA or DNA samples.
Automatically generated - may contain errors
4 protocols using iso seq express kit smrt bell express template prep kit 2
1
Iso-Seq for Long-Read Transcriptome Analysis
2
Jurkat Transcriptome Profiling via PacBio Iso-Seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PacBio (Iso-Seq) data was collected on the Jurkat T-lymphocyte cell line. Jurkat RNA was procured from Ambion (Thermo, PN AM7858). The RNA was analyzed on a Thermo Nanodrop UV-Vis and an Agilent Bioanalyzer to confirm the nominal concentration and ensure RNA integrity. We observed a RIN value of 9.9. From the RNA, cDNA was synthesized using the NEB Single Cell/Low Input cDNA Synthesis and Amplification Module (New England Biolabs).
Approximately 300 ng of Jurkat cDNA was converted into a SMRTbell library using the Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 (Pacific Biosciences). This protocol employs bead-based size selection to remove low mass cDNA, specifically using an 86:100 bead-to-sample ratio (Pronex Beads, Promega). Library preparations were performed in technical duplicate. We sequenced each library on a SMRT cell on the Sequel II system using polymerase v2.1 with a loading concentration of 85pM. A 2-h extension and 30-h movie collection time was used for data collection. The “ccs” command from the PacBio SMRTLink suite (SMRTLink version 9) was used to convert Raw reads (~ 6 million, over 349 Gbps) into Circular Consensus Sequence (CCS) reads. CCS reads with a minimum of three full passes and a 99% minimum predicted accuracy (QV20) were kept for further analysis.
Approximately 300 ng of Jurkat cDNA was converted into a SMRTbell library using the Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 (Pacific Biosciences). This protocol employs bead-based size selection to remove low mass cDNA, specifically using an 86:100 bead-to-sample ratio (Pronex Beads, Promega). Library preparations were performed in technical duplicate. We sequenced each library on a SMRT cell on the Sequel II system using polymerase v2.1 with a loading concentration of 85pM. A 2-h extension and 30-h movie collection time was used for data collection. The “ccs” command from the PacBio SMRTLink suite (SMRTLink version 9) was used to convert Raw reads (~ 6 million, over 349 Gbps) into Circular Consensus Sequence (CCS) reads. CCS reads with a minimum of three full passes and a 99% minimum predicted accuracy (QV20) were kept for further analysis.
3
PacBio Iso-Seq Transcriptome Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PacBio (Iso-Seq) data were collected on the extracted total RNA collected from the HUVEC cell pellet. HUVEC RNA was analysed on an Agilent Bioanalyzer to confirm concentration and RNA integrity for downstream analysis. We observed a RIN value of 10. From this RNA, cDNA was synthesized using the NEB Single Cell/Low Input cDNA Synthesis and Amplification Module (New England Biolabs).
Approximately 200 ng of HUVEC cDNA was converted into a SMRTbell library for usage with the Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 (Pacific Biosciences). Through this protocol, bead-based size selection occurs in order to remove low mass cDNA (less than 500 kb). Each SMRTBell library was sequenced on the SMRT cell on Sequel II system. A 2-hour extension and 3-hour movie collection time was used for data collection. The ‘ccs’ command from the PacBio SMRTLink suite (SMRTLink version 9) was used to convert raw reads into Circular Consensus (CCS) reads.
Approximately 200 ng of HUVEC cDNA was converted into a SMRTbell library for usage with the Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 (Pacific Biosciences). Through this protocol, bead-based size selection occurs in order to remove low mass cDNA (less than 500 kb). Each SMRTBell library was sequenced on the SMRT cell on Sequel II system. A 2-hour extension and 3-hour movie collection time was used for data collection. The ‘ccs’ command from the PacBio SMRTLink suite (SMRTLink version 9) was used to convert raw reads into Circular Consensus (CCS) reads.
4
Single-cell Transcriptome Profiling with PacBio Iso-Seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PacBio Iso-Seq datasets PacBio lrRNA-seq data (i.e., Iso-Seq) was collected on both Jurkat and WTC-11 cell lines. Jurkat RNA was procured from Ambion (Thermo, PN AM7858) and WTC-11 RNA was extracted from WTC-11 cells (Coriell, GM25256). The RNA was analyzed on a Thermo Nanodrop UV-Vis and an Agilent Bioanalyzer to confirm the RNA concentration and ensure RNA integrity. From the RNA, cDNA was synthesised using the NEB Single Cell/Low Input cDNA Synthesis and Amplification Module (New England Biolabs).
Approximately 300 ng of Jurkat cDNA or WTC-11 cDNA was converted into a SMRTbell library using the Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 (Pacific Biosciences). This protocol employs bead-based size selection to remove low mass cDNA, specifically using an 86:100 bead-to-sample ratio (Pronex Beads, Promega). Library preparations were performed in technical duplicate. We sequenced each library on a SMRT cell on the Sequel II system using polymerase v2.1 with a loading concentration of 85 pM. A two-hour extension and 30 hour movie collection time was used for data collection. The `ccsc ommand from the PacBio SMRTLink suite (SMRTLink version 9) was used to convert raw reads into Circular Consensus Sequence (CCS) reads. CCS reads with a minimum of three full passes and a 99% minimum predicted accuracy (QV20) were kept for further analysis.
Approximately 300 ng of Jurkat cDNA or WTC-11 cDNA was converted into a SMRTbell library using the Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 (Pacific Biosciences). This protocol employs bead-based size selection to remove low mass cDNA, specifically using an 86:100 bead-to-sample ratio (Pronex Beads, Promega). Library preparations were performed in technical duplicate. We sequenced each library on a SMRT cell on the Sequel II system using polymerase v2.1 with a loading concentration of 85 pM. A two-hour extension and 30 hour movie collection time was used for data collection. The `ccsc ommand from the PacBio SMRTLink suite (SMRTLink version 9) was used to convert raw reads into Circular Consensus Sequence (CCS) reads. CCS reads with a minimum of three full passes and a 99% minimum predicted accuracy (QV20) were kept for further analysis.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!