RNA-seq Pacbio ISO-seq prepared with 10X 3' of homo sapiens: PBMCs
3.2 ng of cDNA was amplified in a 100 l MAS-PCR reaction using the following reaction conditions: 50 l of MAS PCR mix, 10 l of custom indexed Ai/Qi MAS primers (see Supplementary Table 8 for MAS primers; to enable multiplexing), 15 ng of cDNA library, adding H2O to bring final volume to 100 ul. Reactions were amplified using the following cycling conditions: 98C for 3 minutes, followed by 8 cycles of 98C for 20 s, 68C for 30 s and 72C for 8 minutes, followed by a final 72C extension for 10 minutes. The amplified libraries were cleaned using a 0.8 SPRIselect beads (Beckman Coulter B23318), eluted into 60 l elution buffer, and quantified with Qubit (Thermo Fisher Scientific, Q32851). Barbell formation was carried out by mixing 500 ng of MAS PCR product in 55 l volume with 2 l MAS enzyme and incubated at 37C for 30 minutes. Subsequently, 3ul of barcode MAS adapter and 20 l of MAS ligation additive was mixed into the sample followed by 10 l of MAS ligase buffer and 10 l fo MAS ligase. Sample was thoroughly mixed by gentle pipetting using a wide bore pipette tip to avoid bubble formation. MAS array formation reactions were incubated at 42C for 60 minutes followed by 4C hold (52C lid). Array formation reactions were cleaned using 0.8 SPRIselect beads (Beckman Coulter B23318), eluted into 43 l elution buffer and quantified with Qubit (Thermo Fisher Scientific, Q32851). DNA Damage Repair was carried out by mixing 42 l of sample with 6 l of Repair buffer and 2 l of DNA repair mix, and incubated at 37C for 30 minutes (lid 47C). DNA damage repair reactions were cleaned using 0.8 SPRIselect beads (Beckman Coulter B23318), eluted into 40 l elution buffer and quantified with Qubit (Thermo Fisher Scientific, Q32851). Nuclease treatment was carried out by mixing 40 l of DNA damage repaired library with 5 l Nucelase buffer and 5 l Nuclease mix and incubated at 37C for 60 minutes followed by 4C hold (lid 47C). The final MAS array libraries were cleaned using 0.8 SPRIselect beads (Beckman Coulter B23318), eluted into 20 l elution buffer and quantified with Qubit (Thermo Fisher Scientific, Q32851). Library size was obtained using a Genomic DNA ScreenTape and 4150 Tapestation (Agilent Technologies). Multiplexed ISO-seq libraries were pooled in equimolar amounts and sequenced on two Revio SMRT cells (PN: 102-202-200).