Reagent Preparation
This protocol takes 10 – 12 hrs to complete.Ideally, master mixes for the various steps in the process should be prepared as required.
For maximum stability and shelf-life, enzymes and reaction buffers are supplied separately in the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) Globin. For a streamlined “with-bead”protocol, a reagent master mix with a minimum of 10% excess is prepared for each of these enzymatic steps, as outlined in Tables 2 – 9.
Volumes of additional reagents required for the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) Globin protocol are listed in Table 10.
In some cases, master mixes may be constituted with varying proportions of the total final water requirement. In the examples given in the tables below, all of the required water is included in each master mix, allowing the entire reaction mix to be added in a single pipetting step.
At the safe stopping point at A-tailing, a portion of the water and reaction buffer are added to the beads for storage at 2°C to 8°C for ≤24 hrs.To resume library construction, prepare the master mix with the remaining volume of water and reaction buffer, and the required volume of enzyme. Recommendations on how to formulate the master mix after this safe stopping point are provided in Table 7B.
Always ensure that the reagents required for oligo hybridization, rRNA/globin mRNA depletion, DNase digestion and the PEG/NaCl Solution are fully equilibrated to room temperature before use.
Table 2. Oligo hybridization
*If also processing non blood-derived samples, or if following the KAPA RiboErase (HMR) workflow, the volume of Globin Hybridization Oligos (HMR) must be replaced with an equal volume of RNase-free water (1 μL). For more information refer to the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) technical data sheet – KR1151 v5.17 (or later).
Table 3. rRNA/globin mRNA depletion
Table 4. DNase digestion
Table 5. 1st strand synthesis
Table 6. 2nd strand synthesis and marking
Table 7A. A-tailing (uninterrupted protocol)
Table 7B. A-tailing (safe stopping point)
Table 8. Adapter ligation
Table 9. Library amplification
Table 10. Volumes of additional reagents required
DNase Digestion Cleanup2nd Strand Synthesis and Marking Cleanup1st Post-ligation Cleanup2nd Post-ligation CleanupLibrary Amplification Cleanup