After adding 37.5 µL ProNex beads (Promega, NG2002) to each sample, up to 16 samples were multiplexed. The cDNA library was purified according to the manufacturer’s instructions, eluted into 42 µL 10 mM Tris-HCl, pH 7.4, then re-purified using ProNex beads (1.5:1 vol/vol ratio of beads to sample) and eluted into 45 µL of 10 mM Tris-HCl, pH 7.4. The library was reamplified by preparing PCR reaction mix (20 µL of cDNA library, 25 µL of KAPA HiFi HotStart Uracil+ ReadyMix, and 2.5 µL of 10 µM each PCR primer 1 forward/reverse), and the mixture was kept at 98 ˚C for 3 minutes; 98 °C for 20 seconds, 67 °C for 15 seconds, and 72 °C for 6 minutes (4–6 cycles (see beelow)); and 72 ˚C for 5 minutes; and the mixture was then held at 4 °C. The number of PCR cycles for each amplification was determined by a pilot experiment using quantitative PCR (qPCR) to ensure that the amplification was at the early linear phase. The amplified cDNA library was purified using ProNex beads, as above, and eluted into 26 µL of 10 mM Tris-HCl, pH 7.4. The purified cDNA library was quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32851).
Pronex beads
Manufactured by Promega
Sourced in United States
ProNex beads are magnetic beads used for nucleic acid purification in molecular biology applications. They provide a platform for efficient capture, washing, and elution of target nucleic acids from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (6 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Iso seq express oligo kit, by Pacific Biosciences (2 mentions)
Iso seq express kit smrt bell express template prep kit 2, by Pacific Biosciences (2 mentions)
Neb single cell low input cdna synthesis and amplification module, by New England Biolabs (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Smrtbell express template prep kit 2, by Pacific Biosciences (2 mentions)
Eb buffer, by Qiagen (1 mentions)
Qubit 2.0 system, by Thermo Fisher Scientific (1 mentions)
Smarter pcr cdna synthesis kit, by Takara Bio (1 mentions)
Agilent 2100 system, by Agilent Technologies (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nebnext single cell low input cdna synthesis amplification module, by New England Biolabs (1 mentions)
Sequel system, by Pacific Biosciences (1 mentions)
Ribo zero gold kit, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Genemapper 4, by Thermo Fisher Scientific (1 mentions)
Smrt cell 8m, by Pacific Biosciences (1 mentions)
Internal control 1, by Pacific Biosciences (1 mentions)
10 protocols using pronex beads
1
Next-Generation cDNA Library Preparation
2
Profiling T-cell Receptor Diversity
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Next generation sequencing of the T-cell receptor (TCR) was carried out as previously described [18 (link)]. Briefly, DNA was extracted from patient blood samples using DNeasy Blood & Tissue kit (Qiagen), quantified using a Qubit Fluorometer (ThermoFisher Scientific) and amplified by multiplex-PCR of rearranged variable, diverse, joining (VDJ) segments of the TCR genes, which encode the hypervariable CDR3 domain. The products were size selected using Pronex beads (Promega) and subsequently sequenced on a MiSeq (Illumina).
Analysis of the raw TCR sequences was performed using MiXCR [19 (link)]. A built-in library of reference germline V, D, J, and C gene loci from the ImMunoGeneTics (IMGT) database (imgt.org) is employed by MiXCR. The IMGT nomenclature for TCR gene segments is used throughout the study.
Analysis of the raw TCR sequences was performed using MiXCR [19 (link)]. A built-in library of reference germline V, D, J, and C gene loci from the ImMunoGeneTics (IMGT) database (imgt.org) is employed by MiXCR. The IMGT nomenclature for TCR gene segments is used throughout the study.
3
Iso-Seq Express cDNA Synthesis Protocol
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Briefly, 300 ng of total RNA was reverse-transcribed using the NEBNext Single Cell/Low Input Kit for cDNA synthesis following the steps outlined within Iso-Seq Express Template Procedure & Checklist [PN 101-763-800 version 02 (October 2019)]. The resulting cDNA was purified and size-selected at >1.0 kb using 1.0× ProNex beads (Promega, Madison, WI) with a final elution of 17 μl in buffer EB (elution buffer) (QIAGEN, Hilden, Germany). Subsequent PCR amplification used Takara PrimeStar GXL DNA polymerase (2.50 U total) with NEBNext Single Cell (catalog no. E6421S) and Iso-Seq Express (PN no. 101-737-500) cDNA primers; primer binding site incorporation occurred during first-strand cDNA synthesis. Sample cDNA amplification occurred in duplicate in 50 μl of PCR reactions with each containing 8 μl of purified first-strand cDNA template, 1× PrimeSTAR GXL buffer, 0.1 mM deoxynucleotide triphosphates, 1.25 U of PrimeSTAR GXL DNA polymerase (1.25 U/μl), and 1 μl of NEBNext Single Cell and Iso-Seq Express cDNA PCR primers. PCR cycling conditions were as follows: 30 s at 98°C, 15 cycles (10 s at 98°C, 15 s at 65°C, and 10 min at 68°C), and 5 min at 68°C.
4
Full-length mRNA Sequencing with PacBio
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PolyA-containing mRNAs were enriched with oligo (dT) bead primers. The enriched mRNAs were reverse transcribed into cDNA using a Clontech SMARTer™ PCR cDNA Synthesis Kit (Clontech, CA, USA). Subsequently, the synthesized full-length cDNA was amplified via PCR. The cDNA fragments were purified using Pronex beads (Promega), with ratios varying according to transcript size. Purified cDNA was subjected to DNA damage repair, end repair, and ligation with SMRT dumbbell-type sequencing adapters. Following library construction, the Qubit 2.0 system (Life Technologies) was used for quantification, and the Agilent 2100 system (Agilent Technologies) was used to verify library insert size. The SMRTbell template was annealed with a sequencing primer, bound to polymerase, and sequenced using the PacBio Sequel II platform for data acquisition.
5
Isoform Sequencing of D. magna
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The male and female samples from D. magna were prepared for long-read isoform sequencing (Iso-Seq). Three biological replicates for each sample were conducted. The full-length cDNA was produced from 300 ng of total RNA for each sample using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (New England Biolabs, MA USA) and Iso-Seq Express Oligo kit (Pacific Biosciences, CA USA) according to manufacturer’s protocols. Additionally, ribosomal RNA (rRNA) was removed from 12 μg of total RNA of each sample using the Ribo-Zero Gold kit (Illumina, CA USA). The rRNA-depleted RNA samples were then polyadenylated using the SMARTer smRNA-Seq kit for Illumina (Clontech Laboratories, Inc., CA USA). The full-length cDNA was generated following the same protocols as those used for total RNA. After size selection with the ProNex beads (Promega, WI USA), the full-length cDNA libraries were constructed with the SMRTbell Express kit 2.0 (Pacific Biosciences, CA USA). The concentration and the size distribution of each library were measured using the Qubit 4 Fluorometer (Thermo Fisher Scientific, MA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), respectively. Each library was then sequenced on the Sequel system (Pacific Biosciences, CA USA) using one SMRT Cell 1 M v3 LR with Binding Kit 3.0 and Sequencing Kit 3.0, with a collection time of 20 h.
6
Jurkat Transcriptome Profiling via PacBio Iso-Seq
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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.
7
TCR Repertoire Analysis in Thymic Cells
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Next-generation sequencing of TCR was carried out as previously described [12 (link)]. We obtained thymic CD4+SP cells from mice in different groups using MACS. Briefly, DNA was extracted from these thymic CD4+ SP cells using DNeasy Blood &Tissue kit (Qiagen), quantified using a Qubit Fluorometer (Thermo) and amplified by multiplex-PCR of rearranged variable, diverse, joining (VDJ) segments of the TCR genes, which encode the hypervariable CDR3 domain. The products were size selected using Pronex beads (Promega) and subsequently sequenced on a MiSeq (Illumina). The length and polymorphism of CDR3 were analyzed with GeneMapper 4.1 software (Thermo).
8
PacBio Sequel II CCS Sequencing
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Sequencing libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, #100-938-900) according to the manufacturer's protocol. Briefly, 250 ng of DNA was damage-repaired by mixing it with 7 μl DNA prep buffer, 0.6 μl NAD, and 2 μl DNA Damage Repair Mix v2. This reaction was incubated at 37°C for 30 min before proceeding to the end-repair step. For the end-repair step, 3 μl End Prep Mix was added to the reaction and incubated at 20°C for 30 min, followed by a second incubation at 65°C for 30 min. The reaction was then mixed with 3 μl barcoded overhang adapter, 30 μl Ligation Mix, 1 μl Ligation Enhancer, and 1 μl Ligation Additive to ligate the barcoded adapter. This reaction was incubated at 20°C for 1 h. The SMRTbell libraries were cleaned up with ProNex beads (Promega, #NG2001), pooled, and sequenced on a SMRT Cell 8M on the Pacific Biosciences Sequel II in CCS mode with the following parameters: –noPolish –minLength 10 –minPasses = 3).
9
Iso-Seq library preparation and sequencing
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First-strand cDNA synthesis and cDNA amplification were performed with a NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module (New England Biolabs, MA, USA) and Iso-Seq Express Oligo Kit (Pacific Biosciences (PacBio), CA, USA) according to the procedure and checklist (i.e., Iso-Seq Express Template Preparation for Sequel and Sequel II Systems) provided by PacBio. Amplified cDNA samples were purified with ProNex beads (Promega, WI, USA) using the standard workflow and assuming that most transcripts are ~2 kb. Quantification was performed using a Qubit Fluorometer (Thermo Fisher Scientific) and size distribution was confirmed by a Femto Pulse System.
The Iso-Seq library was prepared by going through the steps of DNA damage repair, end repair and A-tailing, overhang adopter ligation, and cleanup using a SMRTbell Express Template Prep Kit 2.0 (PacBio). Sequencing primer was annealed and polymerase binding, complex cleanup, and sample loading were performed with a Sequel II binding kit 2.1 and internal control 1.0 (PacBio) following the manufacturer’s protocol, and sequencing was performed using the Sequel IIe system. Using one SMRTcell per sample, movie runtime was set to 24 hours for each SMRTcell.
The Iso-Seq library was prepared by going through the steps of DNA damage repair, end repair and A-tailing, overhang adopter ligation, and cleanup using a SMRTbell Express Template Prep Kit 2.0 (PacBio). Sequencing primer was annealed and polymerase binding, complex cleanup, and sample loading were performed with a Sequel II binding kit 2.1 and internal control 1.0 (PacBio) following the manufacturer’s protocol, and sequencing was performed using the Sequel IIe system. Using one SMRTcell per sample, movie runtime was set to 24 hours for each SMRTcell.
10
Single-cell Transcriptome Profiling with PacBio Iso-Seq
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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.
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