*Steps using bioanalyzer can be supplemented with a tapestation protocol
8.1. Picogreen the plate. Note: Use standards starting at 50ng/ul (18 or 14 cDNA PCR cycles) or at 25ng/ul (10 cDNA PCR cycles). If multiple plates, only analyze standards on one plate.
8.2. Run picogreen plate and save values.
8.3. Note: If all samples have expected concentrations on picogreen, continue to 10.5.However, if a sample looks low on PicoGreen STOP and perform the following:
a. Run the final library on Qubit to verify the PicoGreen results.
b. If concentration is good, replace the concentration in the picogreen Excel file and continue to step 10.5.
c. If concentration is low, determine if the RNA was previously run on a BA. If the RNA quality is good, proceed to d.
If the RNA is poor, stop here and notify sequencing facility or troubleshoot before continuing.
d. Obtain the amplified cDNA and run a Qubit with this sample as well as the UHR control and 1-2 other random samples that had good quality picogreen results. Continue to e.
e. Determine if the cDNA levels indicate a successful library was produced.
f. If the cDNA Qubit results are similar to other samples, this indicates a library was generated.Verify this by running the cDNA on a high-sensitivity BA chip. If a good library is seen on the BA, repeat steps 8-10.4 on this sample(s).
g. If the cDNA Qubit results are poor, indicating no library was produced, continue to h.
h. Obtain the original RNA if no previous BA has been run on this sample.Run sample on an appropriate RNA BA chip and inspect RNA quality.
i. If RNA quality is poor indicating degradation, stop here and troubleshoot before continuing.
j. If RNA quality is good, repeat library prep for this sample(s).Note: If samples require a reprep, STOP and wait until other TagSeq jobs for upcoming NovaSeq run are completed.Highlight problem samples in NovaSeq Google Doc. Perform repreps from all jobs in a single batch. Once repreps have acceptable picogreen results, continue to step 10.5, and pool samples with appropriate job.
1.2.Dilute a subset of the samples to approximately 2-3ng/ul each (if <2ng/ul it may be hard to see clearly on BA) and run a single High Sensitivity Bioanalyzer chip to check library quality and concentration.