High throughput library construction method for small RNA of prokaryote
A construction method and prokaryotic technology are applied in the field of high-throughput sequencing library construction of prokaryotic small RNAs, which can solve the problem of no kits, and achieve the effect of reducing purification operations and reducing the probability of RNA degradation.
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[0027] Example 1 Construction of fifteen anaerobic bacteria prokaryotic small RNA strand specific library
[0028] 1. Removal of DNA in total RNA
[0029] 1. Use the following 28μl reaction system for DNA removal reaction:
[0030]
[0031] 2. Mix well, centrifuge briefly and incubate at 37°C for 15 minutes.
[0032] 2. Removal of rRNA in RNA samples after DNA removal
[0033] Use Illumina's Ribo-Zero kit to remove rRNA, the steps are as follows:
[0034] 1.RiboZero TM Preparation of magnetic beads
[0035] A. Vortex and mix the magnetic beads that have been equilibrated at room temperature for 30 minutes. Take 225μl to a low-adsorption RNase-free 1.5ml centrifuge tube and place it on a magnetic stand for 2 minutes until the supernatant is clear. Remove the supernatant.
[0036] B. Remove the centrifuge tube from the magnetic stand, add 225μL of RNase-free water that has been equilibrated at room temperature, pipette at the bottom of the tube ten times to mix, place on the magnetic stand...
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[0110] Example 2 Construction of three microRNA high-throughput sequencing libraries of Mycobacterium smegmatis
[0111] 1. Removal of DNA in total RNA
[0112] 1. Use the following 28μl reaction system for DNA removal reaction:
[0113]
[0114] 2. Purge and mix the above 28μl reaction system, centrifuge briefly and incubate at 37°C for 15min.
[0115] 2. Removal of rRNA in RNA samples after DNA removal
[0116] Use Illumina's Ribo-Zero kit to remove rRNA, the steps are as follows:
[0117] 1.RiboZero TM Preparation of magnetic beads
[0118] A. Vortex and mix the magnetic beads that have been equilibrated at room temperature for 30 minutes. Take 225μl to a low-adsorption RNase-free 1.5ml centrifuge tube and place it on a magnetic stand for 2 minutes until the supernatant is clear. Remove the supernatant.
[0119] B. Remove the centrifuge tube from the magnetic stand, add 225μL of RNase-free water that has been equilibrated at room temperature, pipette at the bottom of the tube ten time...
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