Recommended Notes Before Starting ImmunoPrism Library Preparation

PLEASE READ THE ENTIRE GUIDE BEFORE STARTING LIBRARY PREPARATION

If you have any questions, please do not hesitate to reach out to us at:

support@cofactorgenomics.com

• Use of the included ImmunoPrism Human Control RNA is strongly recommended. This control provides a means to evaluate performance throughout the entire ImmunoPrism process, from library preparation to analysis.

• Keep reagents frozen or on ice unless otherwise specified. Do not use reagents until they are completely thawed. Be sure to thoroughly mix all reagents before use. Keep enzymes at -20 °C until ready to use. Return to -20 °C promptly after use.

• During pre-capture library steps, if samples are not undergoing a specific thermal cycler program, they should be kept cold (CoolRack or similar).  For Part II (Step 12) onwards, the samples should be kept at room temperature.

• Use only pure nuclease-free water. Do not use DEPC-treated water.

• During bead purifications,use freshly made 80% ethanol solutions from molecular grade ethanol. Using ethanol solutions that are not fresh may result in lower yields.

• When pipetting to mix, gently aspirate and dispense at least 50% of the total volume until the solutions are well mixed.

• There are separate protocols for intact samples and degraded samples, for samples from FFPE or samples with RIN <2, we recommend using the degraded sample workflow - there is NO fragmentation step with the lower RIN samples.

• It is important to use the exact amount of starting RNA when moving into library preparation 40ng for degraded samples and 20ng for intact samples.

• Dilutions or concentrations may be needed to start the protocol - all samples move into the workflow with 5uL of total volume

• During Step 7 (adapter ligation), it is important to keep the adapter cold (-20C) in order to avoid adapter dimers

• Additionally, for Step 7, thorough mixing is important due to the viscosity of the ligation master mix

• For all bead cleanup steps, it is important not to disturb the beads with the pipet

• Bioanalyzer traces of the pre-capture libraries may show a peak around 128bp that indicates adapter dimerization, if you observe a peak here, please reach out to us for a review of the QC information

• FFPE or degraded samples received additional PCR amplication cycles (15 rather than 10) because these samples will yield a lower amount of useable RNA

• During Step 9, it is critical to avoid any cross contamination of the individual sample indexes - this would assign reads to the wrong sample and severely impair the statistical power of ImmunoPrism.  Spinning down the individual index tubes to remove any liquid in their caps is recommended before opening the tubes

• It is recommended (especially for the first batch of samples processed in your laboratory) to review the QC for the pre capture libraries with Cofactor following Step 11

• During Step 16. Bind Hybridized Target to the Streptavidin Beads, be sure to vortex every 10-12 minutes to increase the bead capture efficiency. Carefully hold the caps of the warm strip tubes when vortexing to prevent tubes from opening.

• When removing the strip tubes from the thermal cycler for mixing via vortex, be careful to avoid the caps popping off by holding down the caps during the time they are mixing (alternatively you may pipet the samples up and down several times)

• It is critical to perform Step 17. Wash Streptavidin Beads to Remove Unbound DNA as it is written in the protocol to avoid high nonspecific contamination. Be sure to completely resuspend the beads at each wash, completely remove the wash buffers, and move the samples to a fresh strip tube (Step 17.16). Ensure that the streptavidin beads are completely resuspended and remain in suspension during the entire incubation. Splashing on the tube caps will not negatively impact the capture.

• Do not let the streptavidin beads dry out. If needed extend incubation times to avoid drying the beads. If using more than one strip tube work with one strip tube at a time for each wash to avoid drying the beads or rushing, resulting in poor resuspension or other sub-optimal techniques. For first time users, we do not recommend processing more than 8 library reactions at a time.

• If final libraries yield a concentration less than 2 nM, there are still options for sequencing, we would be happy to discuss with you, though Illumina's support will also be knowledgable.