Modification no. | Page and Step in Fluidigm C1 manual | Modification | Reason |
---|---|---|---|
1 | Page 41–43, Pool and Cleanup | Purify each of the Nextera XT reactions individually (not as a single pool) and Elute each individual reaction in 17 μl of Low TE (10:0.1) and quantify each with PicoGreen | Individual purification, elution and quantification of Nextera XT libraries allows for the exclusion of failed sequencing library preps in the final RNA-seq pool |
2 | Page 43, Repeat Cleanup Step | Pool the samples; note the starting volume of the pool. Perform cleanup using AMPure XP beads and elute with the same volume as used when pooled | A pool is generated from 3 ng from each individual library. The library should not include libraries that failed amplification |