GSM7430892: Amyloid Body ePAT 28S-1; Homo sapiens; OTHER
Amyloid-bodies were purified and then RNA was extracted using TRIzol. 1ug of RNA and 1uL of Reverse-ePAT anchored (100uM) primer were heated at 80°C for 5 min. Samples were cooled to room temperature, incubated with 4uL dH2O. 4uL SuperScript IV Reverse Transcriptase Buffer (Life Technologies), 1uL 100mM DTT, 1uL 10mM dTNPs, 1ul RNaseOUT (Life Technologies) 1uL Klenow polymerase (NEB). Samples were incubated at 25°C for 1 hr. Polymerase was then inactivated at 80°C for 10 min, followed by cooling to 55°C for 1 min. Heating block was maintained at 55°C and 1uL of  SuperScript IV was added and incubated for 1 hr then inactivated at 80°C for 10min. ePAT reactions, ePAT reactions were done with Barcoded Reverse-ePAT anchored primer and PCR amplified with Barcoded forward and reverse primers. Reactions were PCR cleaned using NEB PCR purification kit. Samples were prepared for Pacbio sequencing (SMRTbell Template Prep Kit) ePAT followed by PacBio sequencing