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cDNA amplification and capture for PacBio library construction

ZS Zhuo-Xing Shi
ZC Zhi-Chao Chen
JZ Jia-Yong Zhong
KH Kun-Hua Hu
YZ Ying-Feng Zheng
YC Ying Chen
SX Shang-Qian Xie
XB Xiao-Chen Bo
FL Feng Luo
CT Chong Tang
CX Chuan-Le Xiao
YL Yi-Zhi Liu
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Eighty nanograms of cDNA products were amplified using five PCR cycles by using KAPA HiFi HotStart Uracil 2 x ReadyMix (Kapa Biosystems) as well as designed PCR primers containing deoxyuracil, one of which was biotinylated.

Forward primer: 5′-ACTAGUAAGCAGTGGTATCAACGCAGAG −3′

Reverse primer: 5′-Biotin-ACTAGUCTACACGACGCTCTTCCGATCT-3′

The PCR products were then purified using 0.8 volumes of Agencourt AMPure XP Beads (Beckman Coulter), quantified using Qubit dsDNA HS Assay Kits (Thermo Fisher), and assessed via Agilent 2100 DNA HS Assays (Supplementary Fig. 4). The barcode-UMI-poly (dT)-flanked cDNAs were captured on streptavidin-coated M-280 Dynabeads using DynabeadsTM kilobaseBINDERTM Kits (60101, Invitrogen, Carlsbad, CA), whereas the unbound cDNAs were removed.

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