CRC3 Control
CAGE libraries were generated from CRC organoid RNA samples following the protocol by Murata, M. et al. 201428. To increase multiplexing capacity and demultiplexing efficient of libraries sequenced on Illumina 2-color instruments (e.g. NextSeq/NovaSeq), the sequencing adaptors listed in the above protocol were modified to utilise standard Illumina barcoding/indexing strategy (instead of the legacy 3-bp barcode in the beginning of read 1), in this case 8-bp TruSeq Unique Dual Indexes (UDI). To attach the Illumina P5/P7 flow cell adapters and I5/I7 indexes, additional amplification step of 5-6 PCR cycles was performed using the NEBNext UltraTM II Q5 Master Mix (NEB). The PCR reactions were purified with 1.4X AMPure XP beads (Beckman Coulter) to yield the final libraries. Libraries were sequenced (2x50 bp reads) on the NovaSeq6000 v1.5 SP 100 cycles flow cell.