title: GSM5911745: CellSelex_HumanIslet_cycle6; synthetic construct; OTHER
library_construction_protocol: cDNA from cycles 1-8 in the HT “cluster-cell” and cycles M8, M8H1 and M8H2 from the toggle cell SELEX were sampled for HT-sequencing by tagging aptamers' constant region with two sequential PCRs. The first PCR reactions were performed in 100 µl of water containing 1X PCR buffer, MgCl2 solution (1.5 mM), dNTPs (200 µM each), DNA template (5 ng/µl) recombinant Taq polymerase (5 U, Invitrogen) and the PFA and PRA primers corresponding to each cycle described above. The reactions were performed in the GS482 thermocycler (G-STORM) using the following program: 95ºC 5', 5x (95ºC 1', 56ºC 30”, 72ºC 30”), 72ºC 10'. PCR was purified via gel extraction using the QIAquick Gel Extraction Kit (QIAGEN) following manufacturer instructions. The second PCR was performed using the same condition described above but using the UFB and PRB primers  and the following cycles: 95ºC 5', 6x (95ºC 30'', 65ºC 30'', 72ºC 30”), 72ºC 10'. Products were purified by gel extraction, quality evaluated via bio-analyzer (Agilent). Library quantitation and pooling took place at the Hussman Institute for Human Genomics-Center for Genome Technology using the KAPA Library Quantification Kit for Illumina platforms (part# KK4854). 10-13 pM of pooled samples were loaded on the Illumina cBot for cluster generation according to manufacturer's recommendations. Sequencing was performed on an Illumina HiSeq 2000/2500 (HCS 2.0.12.0) using the reagents provided in the Illumina TruSeq PE Cluster Kit v3 and the TruSeq SBS Kit-HS (200 cycle) kit.