Before the tea plant genome sequenced, 20 expressed sequence tags (EST) that annotated as PMEI domain containing proteins were identified from the transcriptome data of the tea plant under CA condition (Wang et al., 2013 (link)). After assembled by Seqman software, a total of 4 contigs were obtained and served as templates to design RT-PCR primers for TA cloning. The TA cloning method was performed as described by Qian et al. (2016) (link). The amplified and purified PCR products were inserted into the pEASY-Blunt Zero vector (TransGen Biotech, Beijing, China), then transferred into Trans5α chemically competent cell (TransGen Biotech, Beijing, China) and sequenced finally. All RT-PCR primers were listed in Supplementary Table 3 .
Trans5α chemically competent cell
Manufactured by Transgene
Sourced in China
Trans5α Chemically Competent Cells are a specialized bacterial strain designed for efficient DNA transformation. They are pre-treated to enhance their ability to take up and incorporate foreign genetic material.
Automatically generated - may contain errors
Lab products found in correlation
Pgem t easy vector, by Promega (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Minibest agarose gel dna extraction kit, by Takara Bio (2 mentions)
Peasy blunt zero vector, by Transgene (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Tianprep mini plasmid kit, by Tiangen Biotech (1 mentions)
Taq plus master mix, by Vazyme (1 mentions)
T vector, by Transgene (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Taq pcr mastermix, by Tiangen Biotech (1 mentions)
Pmdtm19 t vector cloning kit, by Takara Bio (1 mentions)
H dmem, by Thermo Fisher Scientific (1 mentions)
50 bp dna ladder, by Tiangen Biotech (1 mentions)
Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Gel extraction kit, by CWBIO (1 mentions)
Endofree mini plasmid kit 2, by Tiangen Biotech (1 mentions)
Phanta master mix, by Vazyme (1 mentions)
Lipofiter, by Hanbio Biotechnology (1 mentions)
24 protocols using trans5α chemically competent cell
1
Tea Transcriptome PMEI Protein Identification
2
Cloning and Sequencing of SELEX Oligos
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The ssDNA from the last SELEX round (seventh round) were PCR amplified using DreamTaq DNA polymerase (Thermo Scientific) to introduce an A-tail to the PCR products for effective ligation and vector transformation. As depicted in step 12 Supplementary Figure S1 , the dsDNAs were ligated with TOPO vector and cloned into Trans5α chemically competent cell (Transgen Biotech) using Invitrogen’s TOPO-TA Cloning Kit. The bacteria were incubated at 37°C for 15 h on a 50 μg/ml ampicillin containing LB agar plate. Individual colonies (with good shape, see Supplementary Figure S1 ) were picked using pipette tip and added to a premixed polymerase reaction mixture containing Q5® Hot Start High-Fidelity Master Mix, and M13 forward and reverse primers. The mixture was PCR amplified (step 13 Supplementary Figure S1 ) and 2 μl of 6× orange loading dye (NEB) was added to the products and electrophoresed by 2% agarose gel at 120 V for 40 min. Colonies with correct size were picked and dropped (with sterile pipette tips) into 5 ml of 50 μg/ml ampicillin-containing LB medium for replication and growth. Samples are then incubated for 16 h at 250 rpm and at 37°C, TIANprep Mini Plasmid Kit (Tiangen) was used for the plasmid extraction and sent for Sanger Sequencing (Genewiz service was used), see steps 14 and 15 Supplementary Figure S1 .
3
cDNA Amplification and Cloning
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About 2 ng of the pre-amplified cDNA of each cell was further amplified using 2 × Taq Plus Master Mix (Vazyme, Cat. P212), and then cloned into T vector (Transgen, Cat. CT111-01). Next, the ligated plasmid transferred into Trans5α chemically competent cell (Transgen, Cat. CD201-01) by heat shock. The M13 primer were used to identify positive clones inserted with cDNA fragments. Single clones of bacteria were collected for Sanger sequencing to identify the barcode sequence of each cell.
4
Cloning and Transfection of TRIP13 Gene
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The siRNA sequence of TRIP13 gene was synthesized by GenePharma (Shanghai, China) (Table 1 ). The siRNA was dissolved in DEPC water diluted to 20 μM, storing at -20°C for standby use according to the instruction. Human TRIP13 gene ORF cDNA clone expression plasmid was purchased from Sino Biological (Beijing, China), and TRIP13 Gene primer (Supplementary Table 1 ) was purchased from Tianyi Huiyuan (Guangzhou, China). The expression vector was constructed to obtain pcDNA3.1-HA-TRIP13 plasmid and pEGFP-C1-TRIP13 plasmid by Trans5α Chemically Competent Cell (Transgen, Beijing, China) with ampicillin (100 mg/ml) (Maygene, Guangzhou, China) and kanamycin (10 mg/ml) (Maygene, Guangzhou, China), according to Biomiga EZgene™ Plasmid Miniprep Kit (Biomiga, Santiago, CA, USA) instruction and Biomiga EZgene™ PCR/Gel Extraction Kit (Biomiga, Santiago, CA, USA) instruction. We use Lipofectamine 3000 (Invitrogen, Shanghai, China) in accordance with the instruction of manufacturers to configure transfection solution and add fresh medium and mix evenly. The medium was changed after 6 h, and the cells were continued to incubate for 24 h.
5
Cloning and Sequencing of BdOctβR1 in B. dorsalis
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Specific primers (Table 1 ) for nested PCR corresponding to the predicted sequences of BdOctβR1 in B. dorsalis (GenBank: XP_011212557) were designed to obtain the full-length cDNA sequence. PCR was carried out using the high fidelity DNA polymerase with the following procedure: initial denaturation at 98 °C for 2 min, followed by 35 cycles of 98 °C for 15 s, 58 °C for 15 s, and 72 °C for 2 min, and final extension at 72 °C for 10 min. PCR products were separated by electrophoresis on an agarose gel (1.0%). The purified amplicons were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) and transformed into Trans5α chemically competent cell (TransGen Biotech, Beijing, China). The transformants were selected with Luria-Bertani agar plates containing 0.1% ampicillin, and positive clones were sequenced (Invitrogen, Shanghai, China).
6
Purification and Characterization of AFP Protein
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The purified AFP protein (100 μg/ml) was purchased from Fitzgerald (Fitzgerald, MA, USA). Taq PCR MasterMix and 50 bp DNA Ladder were purchased from Tiangen (Tiangen Biotech Co., Ltd., Beijing, China). Dynabeads M-280 Streptavidin was purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA). Bovine serum albumin was purchased from Sigma-Aldrich (Saint Louis, MO, USA). pMDTM 19-T Vector Cloning Kit was purchased from Takara (TaKaRa Biomedicals, Shiga, Japan). Trans5α Chemically Competent Cell was purchased from TransGen (TransGen Biotech Co., Ltd., Beijing, China). All chemicals and reagents used were of analytical grade and prepared with deionized water.
The initial ssDNA library and the primers described inTable 1 were chemically synthesized by Sangon (Sangon Biotech Co., Ltd., Shanghai, China). The ssDNA library was dissolved in the CE Buffer (30 mM NaH2PO4) at a pH of 7.5.
Two human hepatoma cell lines, HepG2 and SMMC7721, and one human lung cancer cell line A549 were purchased from the Shanghai cell bank, Chinese Academy of Sciences. All these cells were cultured in Dulbecco’s modified eagle medium with high glucose (DMEM-H) (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco).
The initial ssDNA library and the primers described in
Two human hepatoma cell lines, HepG2 and SMMC7721, and one human lung cancer cell line A549 were purchased from the Shanghai cell bank, Chinese Academy of Sciences. All these cells were cultured in Dulbecco’s modified eagle medium with high glucose (DMEM-H) (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco).
7
Minigene Approach to Verify Splicing Mutations
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Using the Minigene approach to verify the effect of mutations on gene splicing has been widely accepted (Nakajima et al., 2016 (link)). Fragments of CACNA1F gene spanned intron 30 to intron 35 were amplified from control and patient genomic DNA (I1 and II3) respectively using 2× Phanta® Master Mix (Vazyme, Nanjing, China) and primer 2 h‐CA‐F/R (Table 1 , Figure 3a ). The amplicons were cloned into pcDNA3.1(+) vector at the Nhe I and Xho I sites respectively, then transformed into Trans5α chemically competent cell (TransGen, Beijing, China). Positive clones were analyzed by Sanger sequencing (Figure S4 ). EndoFree Mini Plasmid Kit II (TianGen, Beijing, China) was used to extract the pcDNA3.1‐CA‐wt/mut plasmids. Transient transfection studies were performed in HEK293T cells in 6‐well plates using LipoFiter (HANBIO, Shanghai, China), and total RNA was extracted by RNAsimple Total RNA Kit (TianGen, Beijing, China). Reverse transcription was performed using HiScript®IIQ RT SuperMix for qPCR (Vazyme, Nanjing, China). To analyze the splicing products, PCR was performed using primer 3 h‐CA‐F/R present in exon 31 and 34 (Table 1 , Figure 3a ). Five‐microliter PCR products were investigated by electrophoresis on a 2% agarose gel. Amplified bands were directly excised from the gel and purified using Gel Extraction Kit (CWBIO, Beijing, China), then analyzed by Sanger sequencing.
8
Recombinant Protein Expression and Purification
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All oligonucleotides were purchased from Sangon Biotech (Shanghai, China) and are
shown in Table S1. Bsm DNA Polymerase (Large Fragment), T4 polynucleotide kinase
were purchased from Thermo Scientific. Plasmid pET-28(a) was purchased from
Novagen, now part of Merck Biosciences. Trans5α Chemically Competent
Cell, BL21(DE3) Chemically Competent Cell, FastPfu DNA Polymerase and dNTP were
purchased from TransGen Biotech (Beijing, China). Ni–agarose His tag
protein purification kit was bought from Beijing CoWin Biotech.
[γ-32P]ATP was purchased from Furui Biological
Engineering (Beijing, China).
shown in Table S1. Bsm DNA Polymerase (Large Fragment), T4 polynucleotide kinase
were purchased from Thermo Scientific. Plasmid pET-28(a) was purchased from
Novagen, now part of Merck Biosciences. Trans5α Chemically Competent
Cell, BL21(DE3) Chemically Competent Cell, FastPfu DNA Polymerase and dNTP were
purchased from TransGen Biotech (Beijing, China). Ni–agarose His tag
protein purification kit was bought from Beijing CoWin Biotech.
[γ-32P]ATP was purchased from Furui Biological
Engineering (Beijing, China).
9
DNMT3A Mutation Screening Protocol
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In this study, the mutations in exon 23 as well as adjacent intronic regions were focused, because most of DNMT3A mutations were concentrated in exon 23. The polymerase chain reaction (PCR) mixture was 50 μL, containing 100 ng of genomic DNA, 5 μL of 10 times buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 10 pmol of upstream and downstream primers, and 1 to 2 U of Taq DNA polymerase (Promega, USA). The primers’ sequences could be found in Table 1 . The cycling condition was as follows: 5 minutes at 95 °C for predenaturation, 40 cycles of 30 seconds at 95 °C, 30 seconds at 55 °C, and 30 seconds at 72 °C, and 10 minutes at 72 °C for final extension. The PCR products were sent to Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. and directly sequenced using AB PRISM 3730 Automated Sequencer. Four PCR products with unsatisfactory sequencing results were subcloned into pEASYTM-T5 Zero cloning vector. The recombinant plasmids were transformed into Trans 5α chemically competent cell (Transgene, Beijing, China). Five to 10 clones for each product were selected for plasmid sequencing after incubation at 37 °C for 15 hours.[22 (link)]
10
Flanking erm(X) Determinants by SEFA PCR
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An attempt was made to obtain flanking regions of erm determinants by SEFA PCR (Wang et al. 2007) (link). PCR amplification of the downstream region of erm(X) was performed using three gene-specific primers, ermXF_SP1, ermXF_SP2, and ermXF_SP3 (Table 1), and PCR amplification of the upstream region of erm(X) was performed using three genespecific primers, ermXR_SP1, ermXR_SP2, and ermXR_SP3 (Table 1). The primers for SEFA PCR were designed based on the sequence of erm(X) amplified in this work. SEFA PCR was conducted in accordance with the manufacturer's instructions (Wang et al. 2007) (link), and the amplified PCR products were subsequently gel-purified and cloned using the MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa, Dalian, China), pMD®18-T Vector (TaKaRa, Dalian, China), and Trans 5α Chemically Competent Cell (TransGen, Beijing, China), respectively, according to the kit protocols. All sequencing was performed by BGI (Shenzhen, China).
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