Selection using anti-FLAG M2 antibody
First, 40 µl of Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) were washed
two times with TBS-T buffer (50 mM Tris HCl, pH 7.4, with 150 mM, NaCl,
1 mM EDTA, 0.2% Tween 20), using the DynaMag‐2 magnetic stand (Thermo
Fisher Scientific). A total of 100 ng of mRNA-peptide initial library or
mRNA/cDNA-peptide conjugate initial library samples were diluted with
500 µl of TBS-T buffer. Diluted samples were mixed with washed beads and
incubated at room temperature with gentle mixing in a circular rotator
for 1 hour to allow binding. After the incubation, the supernatant was
removed, and the beads were further washed three times with TBS-T and
additional three times with TBS buffer (without Tween 20). Through 1 to
3 round, beads were resuspended in 50 µl TBS buffer and solutions were
directly applied to the RT-PCR reaction (see next section for detail) to
generate double strand DNA library for the next selection round and
sequencing. For the 4th round samples, we performed elution in a
stepwise manner using FLAG Peptide (Sigma-Aldrich) with increasing
concentrations from 4 μg/ml to 20 μg/ml to 100 μg/ml. Each of the eluted
fractions, along with the remaining beads, were analyzed with RT-PCR.