Question: How are the ratios of SPRIselect beads calculated in 10x assays?
Answer: The calculation of the ratio of SPRIselect beads: DNA (reaction volume) indicated in the User Guide of CG000204 (3’ v3.1) is used to explain how one double-sided size selection is performed in two steps as follows.
During the v3.1 fragmentation reaction, you will:
Therefore, your total post fragmentation, end-repair, and A-tailing volume is 50 μl. Then in step 3.2, you will add 30 μl of SPRIselect beads to the 50 μl reaction.
With the ratio of SPRIselect beads: reaction volume as 0.6X (30:50), DNA fragments larger than 600 bp preferentially bind to SPRIselect beads. After incubation, you will place the reaction tube strip on a magnetic stand to pellet the SPRIselect beads. You will then transfer 75 μl of the supernatant (containing DNA fragments smaller than 600 bp) into a fresh tube and add 10 μl of SPRIselect beads to make the ratio of SPRIselect: reaction volume as 0.8X, calculated as follows.
For note, the volume of supernatant (75 μl) is not used in the calculation. Instead, you will use the total volume of SPRIselect added (30 μl + 10 μl) over the original volume of the sample (50 μl) to achieve a final ratio of 0.8. With 0.8X, the fragments larger than 200 bp preferentially bind to SPRIselect beads. You keep the beads (with the DNA bound) and discard the supernatant, leading to the eluted fragments ranging from 200 to 600 bp.
As another example of how the SPRIselect ratios are calculated, Step 2.3B from Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Surface Protein (User Guide CG000317) requires a 2.1X SPRIselect cleanup.
The volume of post cDNA amplification is 100 μl. In step 2.3a, 60 μl of SPRIselect beads are added to the 100 μl reaction to a total of 160 μl. 75 μl will be carried over into the second SPRI reaction. 75 μl / 160 μl is 47%. This means that 47% of the 60 μl SPRI beads (28 μl) will be in the second SPRI step.
The second SPRI step will have 28 μl SPRI solution from the previous step and (75 μl total - 28 μl SPRI beads) 47 μl of the sample. Then 70 μl SPRI beads were added, bringing the total to 98 μl of SPRI with 47 μl of the sample. This is a 2.1X ratio.
In summary, as illustrated in the following schematic overview (Fig. 1), during 1st SPRI cleanup at 0.6X, SPRI beads selectively bind DNA fragments larger than 600 bp, and the beads are discarded. During the 2nd SPRI cleanup, the supernatant (with DNA fragments smaller than 600 bp) is mixed with a fresh volume of SPRIselect beads to make a SPRIselect: reaction volume ratio of 0.8X. At 0.8X, SPRI beads selectively bind to the DNA fragments larger than 200 bp. At the 2nd SPRI cleanup, the beads are kept, and the DNA is eluted. After two SPRI cleanups, the final sample with a tight fragment size distribution (200 – 600 bp) is achieved.