RNA-seq Pacbio MAS-seq 16-mer prepared with PIP-seq, hashtagged fixation protocol vs fresh panel, of homo sapiens: PBMCs
MAS-seq 16-mer libraries were prepared according to manufacturers instructions (PacBio MAS-seq kit Cat, 102-407-900). Briefly, 55ng cDNA was split between 16 MAS PCR reactions, each with the following final reaction conditions: 50 l of MAS PCR mix, 15 ng of cDNA library, adding H2O to bring final volume to 90 ul, then adding 10 l of of MAS primer mix pairs composed of sequential primers to each respective PCR reaction (see primer table). 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 pooled and cleaned using 0.8 SPRIselect beads (Beckman Coulter B23318), eluted into 50 l elution buffer and quantified with Qubit (Thermo Fisher Scientific, Q32851). MAS array formation was carried out by mixing 4 ug of MAS PCR product in 47 l volume with 10 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 of 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).