Product Advantages
Product Application Efficiently remove rRNA from 10 ng~1000 ng of nematode RNA samples, while maximizing the recovery of other mRNA and non-coding RNA, and increasing the proportion of valid data for subsequent research in related fields such as whole transcriptome sequencing and long non-coding RNA sequencing. Product components  Save Instructions
***RNA samples should be kept on ice prior to hybridization. 1. Vortex the probe mix and centrifuge quickly to collect the droplets on the tube wall to the bottom of the tube. 2. Probe and RNA sample hybridization system configuration: Add 12 μL of RNA sample to a PCR tube or plate. If the sample volume is > 12 μL, adjust the volume of HB accordingly, so that the HB volume is 0.25 times the total volume. The total reaction volume should not exceed 40 μL. Configure the system according to the table below.  *As per manufacturer's instructions Ensure that the enzyme is active at 68°C. RNase preparation can also be added during the preparation of magnetic beads.3. Vortex to mix and centrifuge briefly to collect droplets on the tube wall. 4. Place the mixed sample in a PCR instrument for hybridization reaction. The hybridization reaction program is set as follows:  5. Prepare Streptavidin Magnetic Beads (SMB) 5.1 Vortex the beads to fully suspend them. Do not centrifuge to prevent the beads from settling. 5.2 Transfer 40 μL of the resuspended magnetic beads to a new EP tube for each sample. For cleaning of SMB magnetic beads required for multiple samples, these can be performed together, for example, by aliquoting the magnetic beads for 6 samples (240 μL) or 12 samples (480 μL) into one tube for cleaning. 5.3 Place the tube on the magnetic stand and let it stand until the solution becomes clear (the time here may vary depending on the performance of the magnetic stand, the Thermo magnetic stand (Cat. No.: 12321D) takes about 2 minutes). 5.4 Discard all supernatant. 5.5 Add 80 μL of DB to each sample (e.g., 480 μL for 6 samples, 960 μL for 12 samples) and vortex to mix thoroughly. 5.6 Repeat steps 5.3 to 5.5. 6. rRNA Removal 6.1 Centrifuge the hybridization samples (~20 μL) from step 4, then add 80 μL of the prepared streptavidin magnetic beads (step 5) to each sample and mix thoroughly. 6.2 Incubate at room temperature for 15 minutes, shaking every 5 minutes to mix thoroughly, and then incubate at 50°C in a PCR machine for 5 minutes.  6.3 Immediately after the reaction, place the sample tube on a magnetic stand and let it stand until the solution becomes clear (the time here may vary depending on the performance of the magnetic stand. Thermo magnetic stand ( Item No.: 12321D, about 2 minutes ), carefully transfer the supernatant to a new tube. 6.4 Place the sample on a magnetic stand until the solution becomes clear to remove any remaining magnetic beads (recommended). 6.5 Carefully transfer 96 μL of the supernatant to a new tube (recommended). It is recommended to perform steps 6.4 and 6.5 to remove any traces of magnetic beads that may have been absorbed, but this is not mandatory. At this point, the RNA can be stored at -20°C overnight or at -80°C for one month. ★Safety stopping point 7. RNA Purification and Recovery RNA samples after rRNA depletion must be purified to remove salts and buffer concentrates prior to library preparation. 7.1 Add 180 μL of resuspended purified magnetic beads (PB) (equilibrated at room temperature for 30 min before use) to the sample in step 6 (~100 μL) and mix thoroughly. 7.2. Let stand at room temperature for 5 minutes. 7. 3 Place the sample tube on the magnetic stand and let it stand for 3-5 minutes until the solution becomes completely clear. 7. 4. Discard the supernatant. 7. 5. Keep the sample tube on the magnetic stand and add 200 μL of freshly prepared 80% ethanol (the ethanol liquid surface should cover the magnetic beads. If the liquid surface does not cover the magnetic beads, add more 80% ethanol) and let it stand for 30 seconds. 7. 6. Discard the supernatant and repeat the wash. 7. 7 Centrifuge briefly to remove any remaining wash solution at the bottom of the tube. 7. 8. Place the sample tube on the magnetic rack for 3-10 minutes to allow the magnetic beads to dry naturally (be careful not to crack the surface of the magnetic beads and avoid over-drying, as over-drying will affect the RNA recovery rate). 7. 9 Remove the sample tube from the magnetic stand, add 10-15 μL of RNase-free water, and resuspend and mix. 7. 10 Incubate at room temperature for 5 min. 7. 11 Place the incubated sample tube on the magnetic rack and let it stand for 2-5 minutes until the solution becomes clear. 7. 12 Transfer the supernatant containing RNA to a new tube. 7.13 At this point, store the RNA at -80°C. |
