cDNAs were enriched by PCR for 15 cycles. The objective strips were collected with 2% Certified Low- Range Ultra Agarose (Bio-Rad). Through Picogreen (Invitrogen) technology, RT-activity was evaluated by using TBS380 (Invitrogen). PCR amplification was performed to harvest clusters with the cBot, and the clusters were sequenced by the Hiseq platform (Illumina). The cleaned data were obtained by removing low quality tags, cleaning and selecting sequences according to the length in base pairs and analysed according to the reference genome. The target genes of similarly expressed mRNAs were analysed with expressive pattern cluster analysis.
Tbs380
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TBS380 is a laboratory equipment designed for general use in scientific research and analysis. It functions as a bench-top centrifuge capable of processing a variety of sample types. The device enables the separation of components within liquid samples through the application of centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
Truseq rna sample prep kit, by Illumina (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Certified low range ultra agarose, by Bio-Rad (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Poly t oligo attached magnetic beads, by Thermo Fisher Scientific (1 mentions)
End repair mix, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Nanodrop 2000, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Hiseq xten novaseq 6000 sequencer, by Illumina (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
6 protocols using tbs380
1
Transcriptome Analysis by NGS
2
Hepatopancreas RNA Extraction and Transcriptome Analysis
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Total RNA was extracted from the hepatopancreas using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA quality and quantity were determined using Agilent 2100 and NanoDrop 2000 prior to subsequent experiments. Only high-quality RNA samples (1.8 < optical density [OD]260/280 < 2.2; 28S:18S > 1.0; RNA >5 μg) was used for the transcriptome analysis.
The RNA-Seq transcriptome library was prepared using a Truseq RNA Sample Prep Kit (Illumina). PolyA mRNA was purified using poly-T oligo attached magnetic beads (Invitrogen), and was randomly segmented into 200-bp fragments by fragmentation buffer. Then, first-strand complementary DNA (cDNA) was synthesized using reverse transcriptase and random primers, followed by synthesis of second-strand cDNA. The second-strand cDNA was end-repaired using End Repair Mix (Illumina), and a single A base was added at the 3ʹ end for adapter ligation. The cDNA target fragments were selected on 2% Low Range Ultra Agarose (Bio-Red), followed by 15 cycles of PCR amplification. After TBS-380 (Invitrogen) quantification, bridge PCR was performed to amplify the DNA fragments to single-molecule DNA clusters, which were subsequently used in HiSeq 4000 (Illumina) sequencing.
The RNA-Seq transcriptome library was prepared using a Truseq RNA Sample Prep Kit (Illumina). PolyA mRNA was purified using poly-T oligo attached magnetic beads (Invitrogen), and was randomly segmented into 200-bp fragments by fragmentation buffer. Then, first-strand complementary DNA (cDNA) was synthesized using reverse transcriptase and random primers, followed by synthesis of second-strand cDNA. The second-strand cDNA was end-repaired using End Repair Mix (Illumina), and a single A base was added at the 3ʹ end for adapter ligation. The cDNA target fragments were selected on 2% Low Range Ultra Agarose (Bio-Red), followed by 15 cycles of PCR amplification. After TBS-380 (Invitrogen) quantification, bridge PCR was performed to amplify the DNA fragments to single-molecule DNA clusters, which were subsequently used in HiSeq 4000 (Illumina) sequencing.
3
RNA-seq Protocol for Biological Replicates
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Three biological replicates were used for each stage, BP, BS, PreHy and Hy. Each replicate was isolated from the mixture of 3 independent single samples. For RNA-seq, the methods were as previously described [46 (link)]. Total RNA was extracted and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA yields were quantified using the NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware). RNA quality was confirmed by agarose gel electrophoresis and RNA integrity number (RIN) was checked by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Sequencing libraries were generated using the Truseq™ RNA Sample Prep Kit (Illumina, San Diego, CA, USA) following manufacturer’s instructions. After quantification by TBS380 (Invitrogen, Carlsbad, CA, USA), paired-end RNA-seq sequencing library was performed the Illumina HiSeq 4000 (2 × 150 bp read length) platform.
4
Phage Genomic DNA Extraction and Sequencing
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The extraction and purification of the genomic DNA of phages were conducted according to the instructions of the Lambda Phage Genomic DNA Kit (ZP317, Zoman Biotechnology Co., Ltd., Beijing, China). Total phage DNA integrity was assessed using 2% agarose gel electrophoresis and quantified with TBS 380 (Invitrogen, Carlsbad, CA, USA). The Illumina NovaSeq 6000 platform was employed for whole-genome sequencing (Illumina, San Diego, CA, USA). The original sequences were trimmed and optimized by the Trimmomatic-0.39 and ABySS software, respectively, and genome assembly and coding gene prediction were performed using the GapCloser (version 1.12) and GeneMarkS software (version 4.28). In addition, the protein functions of the newly determined genes were compared with Basic Local Alignment Search Tool (BLAST) NR, Swiss-Prot, eggNOG, KEGG, and GO databases (comparison criteria: E value ≤ 1e-5). The National Center for Biotechnology Information Search database (NCBI, https://www.ncbi.nlm.nih.gov/) was used for sequence alignment, and MAGA X software and iTOL software (version 2.1) were used to construct and embellish the phylogenetic tree.
5
Transcriptome analysis of Thitarodes xiaojinensis
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Each group, namely, Tx-BP, MOCK1, Tx-BS and MOCK2, contained three biological replicates. Each biological replicate was collected from three individual larvae. For RNA-sequencing, we collected samples from diverse Thitarodes xiaojinensis fat bodies (Tx-BP, MOCK1, Tx-BS and MOCK2) as previously described [30 (link)]. The complementary DNA (cDNA) libraries were constructed and sequenced by Shanghai Majorbio Biopharm Technology Co., Ltd. (Shanghai, China). After quantification by TBS380 (Invitrogen, Carlsbad, CA, USA), the library was sequenced by the Illumina HiSeq xten/NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA) using the paired-end method, 2 × 150 base pair (bp) read length.
6
Small RNA Sequencing Protocol
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Three grams of fresh material was sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) for deep sequencing. Total RNA was spliced at the 5′ and 3′ ends using a TruseqTM Small RNA Sample Prep Kit. First-strand cDNA was obtained via reverse transcription with random primers using the TruseqTM Small RNA sample prep Kit. The library was enriched using PCR for 11 to 12 cycles. The product was recovered using PAGE electrophoresis and quantitated on a TBS380 (Invitrogen, Carlsbad, CA, USA). The clusters were generated using the cBot system and sequenced on an Illumina HiSeq 2000 platform [34 (link)]. The raw sequencing data were deposited in the NCBI Short Read Archive (SRA, BioProject ID: PRJNA673290).
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