C. elegans Ribo-seq rRNA Removal Kit

• The probe hybridization capture method removes the RNA without enzymatic reaction and will not cause non-specific degradation of the RNA of interest;
• The experimental process is simple, the matching is automated, and time is saved;
• Optimized probe design provides comprehensive rRNA coverage with no off-target effects.

It can efficiently remove rRNA and Globin mRNA from 10 ng~1000ng of total RNA samples in human blood, while maximally recovering other mRNA and non-coding RNA, and increase the proportion of effective data for subsequent research in related fields such as whole transcriptome sequencing and long non-coding RNA sequencing.

Product composition


Save Instructions

The reagent kit should be stored at 2-8℃ and the probe should be stored at -20℃.

Procedure

***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. Configuration of probe and RNA sample hybridization system:
Add 12 μL of RNA sample to a PCR tube or PCR plate (containing 10 ng-1000 ng of total RNA, Note: When using the Universal rRNA Removal Kit for Bacteria, Prokaryotes, and Fungi V1 to remove rRNA, the total RNA input amount should not exceed 200 ng, and it is recommended to use 100 ng of total RNA for rRNA removal; when using the Universal rRNA Removal Kit for Bacteria, Prokaryotes, and Fungi V2 to remove rRNA, the total RNA input amount should not exceed 400 ng, and it is recommended to use 200 ng of total RNA for rRNA removal). If the sample volume is > 12 μL, adjust the volume of HB accordingly so that the HB volume is 0.25 × the total volume. The total reaction volume cannot exceed 40 μL. Configure the system according to the following table:

*According to manufacturer's dosage 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 to fully suspend the streptavidin magnetic beads. Do not centrifuge to prevent the magnetic beads from settling.
5.2 Take 40 μL of resuspended magnetic beads for each sample and put them into a new EP tube. For the cleaning of SMB magnetic beads required for multiple samples, they can be done together, such as dispensing the magnetic beads of 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 will vary depending on the performance of the magnetic stand, Thermo magnetic stand (Cat. No.: 12321D), about 2 min).
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) in step 4, 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 min, vortexing every 5 min, and then incubate at 50°C on a PCR machine for 5 min.


6.3 After the reaction is completed, place the sample tube on the magnetic rack and let it stand until the solution becomes clear (the time here will vary due to different magnetic rack performances. Thermo magnetic rack ( Item No.: 12321D, about 2 min ) Carefully transfer the supernatant to a new tube.
6.4 Place the sample on a magnetic stand until the solution becomes clear to remove trace residual 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 possible traces of magnetic beads, but it is not necessary.
At this point, the RNA can be stored at -20°C overnight or at -80°C for a month. ★Safety stop point

7. RNA Purification and Recovery
The rRNA-depleted RNA samples must be purified to remove salts and buffer concentrates prior to library preparation.
7.1 Add 10 μL of 3M sodium acetate (SA) to the sample in step 6 (~100 μL);
7.2 Add 1 μL of acrylamide (LA);
7. 3. Vortex to mix;
7. 4 Added 333 μL of anhydrous ethanol;
7. 5. Vortex to mix;
7. 6 -80℃ for 30min or -20℃ overnight;
7. 7 Centrifuge at 11000g (or highest speed), 4℃ for 30 min;
7. 8 Carefully remove the supernatant, making sure the pipette tip does not touch the precipitate;
7. 9 Add 200 μL 70% ethanol to wash the precipitate;
7. 10 Centrifuge at 11000g (or highest speed) at 4°C for 5 min;
7. 11 Carefully remove the supernatant, making sure the pipette tip does not touch the precipitate;
7. 12 Repeat the ethanol washing step (steps 7.9 to 7.11);
7.13 Dry the precipitate at room temperature for 5 minutes;
7.14 Add 10 μL RNase-free water or other suitable elution buffer to dissolve RNA and store at -80℃.