Genomic DNA (gDNA) was diluted in nuclease-free water and amplified by nested PCR in the 16s rRNA and the rpoB genes per the conditions outlined in Supplementary Table 1
Nucleospin pcr clean up kit
Manufactured by Takara Bio
The NucleoSpin PCR Clean-up Kit is a laboratory equipment product designed for the purification of PCR products. It efficiently removes primers, nucleotides, polymerases, and other impurities from PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (3 mentions)
Platinum taq dna polymerase reaction kit, by Thermo Fisher Scientific (2 mentions)
In fusion hd cloning kit, by Takara Bio (2 mentions)
Smarter race cdna amplification kit, by Takara Bio (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
9 protocols using nucleospin pcr clean up kit
1
Nested PCR Amplification and Sequencing
2
Seamless DNA Assembly Protocol
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PCR was carried out with
KOD One DNA polymerase (TOYOBO) according to the manufacturer’s
instructions. The oligonucleotide primers were designed so that the
DNA fragments to be assembled overlap each other by 50 bp at the ends.
The oligonucleotide primer sequences and combinations of the primers
and templates are listed inTables S1 and S2 . The PCR products were purified using the NucleoSpin PCR clean-up
kit (Takara).
KOD One DNA polymerase (TOYOBO) according to the manufacturer’s
instructions. The oligonucleotide primers were designed so that the
DNA fragments to be assembled overlap each other by 50 bp at the ends.
The oligonucleotide primer sequences and combinations of the primers
and templates are listed in
kit (Takara).
3
High-throughput CRISPR library screening
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The sgRNA regions were first amplified from frozen stocks using round 1 forward and reverse primers (Table S1 ) and then cleaned up using an Exo-CIP PCR cleanup kit (NEB). The PCR products were diluted 1:50 and amplified again using custom primers containing Nextera adapters and indices. Samples were then pooled and cleaned using a NucleoSpin PCR cleanup kit (TaKaRa). Sequencing was performed on an Illumina NextSeq 500 system. Spacer sequences were extracted from fastq files and counted by exact matching to the designed library sequences.
4
Nested PCR Amplification and Sanger Sequencing
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Genomic DNA (extracted as described above) was diluted in nuclease-free water and ampli ed by nested PCR per the conditions outlined in Additional File 1. For each rst-round PCR reaction, 500 ng (5 µL) of gDNA was added into a 50 µL reaction and ampli ed using the Platinum Taq DNA Polymerase reaction kit (Invitrogen, Carlsbad, CA). For nested PCR reactions, 1 µL of the rst-round PCR product was added to a 50 µL reaction containing the nested primers and Platinum Taq DNA Polymerase. Each round of PCR contained a positive control of BCG DNA and a negative no template control. Following nested PCR, each reaction was examined on a 1% agarose gel. Each PCR amplicon showing the correct size was cleaned up using a Nucleospin PCR Clean-up Kit (Takara) and eluted into 30 µL of EB buffer. Puri ed PCR amplicons (20 ng) were sent for Sanger sequencing using both forward and reverse nested primers in separate reactions. Following sequencing, DNA sequence quality was examined in 4Peaks software and low-quality reads from the 5' and 3' ends removed. Consensus sequences generated through the alignment of forward and reverse reads were analyzed using BLAST analysis.
5
Cloning PTPN4 Protein Domain
6
RNA Extraction and Immunoglobulin Gene Sequencing
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Total RNA was extracted from 20 million PBMCs into 30 μl of water with TRIzol Reagent (Life Technologies). The reverse transcription (RT) was performed with SuperScript III (Life Technologies) and oligo(dT)12–18. The cDNA was purified and eluted in 20 μl of elution buffer (NucleoSpin PCR Clean-up Kit, Clontech). The immunoglobulin gene-specific PCRs were performed with Platinum Taq High-Fidelity DNA Polymerase (Life Technologies) in a total volume of 50 μl, with 5 μl of cDNA as template, 1 μl of gene-specific primers and 1 μl of 10 μM reverse primer. The primers each contained an appropriate adaptor sequence (A or trP1) for subsequent PGM sequencing. Two sequencing directions (Fig. S3A ) and their respective primer sets were designed (Tables S1–S3 ). 25 cycles of PCRs were performed and the expected PCR products (~500 bp) were gel purified (Qiagen).
7
Single-Cell B-Cell Receptor Repertoire Analysis
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Sample preparation was done using 5′-rapid amplification of cDNA ends (RACE) PCR with the primers listed in Supplementary Table 9 and reported in our previous studies6 (link),7 (link). Briefly, the total RNA was purified from 5 to 10 million PBMCs into 30 μL of water using RNeasy Mini Kit (Qiagen, Valencia, CA). For the unbiased repertoire analysis, 5′-RACE was performed using SMARTer-RACE cDNA Amplification Kit (Clontech). The cDNA was purified and eluted in 20 μL of elution buffer (NucleoSpin PCR Clean-up Kit, Clontech). The immunoglobulin PCRs were conducted with Platinum Taq High-Fidelity DNA Polymerase (Life Technologies) in a volume of 50 μL, containing 5 μL of cDNA as a template, 1 μL of 5′-RACE primer, and 1 μL of 10 µM reverse primer. To facilitate NGS on the Ion GeneStudio S5 system or the Ion Personal Genome Machine (PGM) system, the forward 5′-RACE primer contained a P1 adaptor, while the reverse primer contained an A adaptor and an Ion XpressTM barcode (Life Technologies) to differentiate the antibody libraries from different time points. A total of 25 cycles of PCRs were performed and the PCR products (~600 bp) were gel purified (Qiagen)6 (link),7 (link).
8
Cloning PTPN4 Protein Domain
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DNA sequence corresponding to amino acids (aa) 611–926 of PTPN4 was amplified via PCR from cDNA clone ID BC010674. PCR product was purified using NucleoSpin PCR clean-up kit (Clontech) and cloned into pET24-based vector using InFusion HD cloning kit (Clontech). Transformations and plasmid isolations were performed using standard protocols. Resulting PTPN4 expression construct contained N-terminal 6× his tag followed by TEV protease cleavage site.
9
Unbiased B cell repertoire analysis
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To ensure the unbiased analysis of the donor B cell repertoires (3 (link), 61 (link), 72 (link)), an improved version of the 5′-RACE PCR protocol for sample preparation has been reported in our recent study (31 (link)). Here, total RNA was extracted from 1 million to 5 million PBMCs into 30 μl of water with RNeasy Mini Kits (Qiagen, Valencia, CA). For unbiased repertoire analysis, 5′-RACE was performed with SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA). The cDNA was purified and eluted in 20 μl of elution buffer (NucleoSpin PCR Clean-up Kit, Clontech). The immunoglobulin PCRs were set up with Platinum Taq High-Fidelity DNA Polymerase (Life Technologies, Carlsbad, CA) in a total volume of 50 μl, with 5 μl of cDNA as template, 1 μl of 5′-RACE primer or gene-specific forward primers, and 1 μl of 10 μM reverse primer. To facilitate NGS on the Ion GeneStudio S5 system, the forward 5′-RACE primer contained a P1 adaptor, while the reverse primer contained an A adaptor and an Ion Xpress barcode (Life Technologies) to differentiate libraries from different time points. A total of 25 cycles of PCRs were performed, and the 5′-RACE PCR products at ~600 base pairs were gel-purified (Qiagen, Valencia, CA).
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