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Pupal wings were dissected from the pupae in PBST, and wing cuticles manually removed with care.
Cells that did not migrate or invade through the pores of the Transwell inserts were manually removed with a cotton swab.
After 3 days of culture, single and/or clones composed of 2 8 cells were manually removed with a fine-tip micropipette and suspended/washed in cold RNase-free 0.01M PBS.
The cells were manually removed with fine forceps and each cell was placed in a cryogenic tube with 350 ml lysis buffer (buffer RLT, Qiagen, Valencia, CA) and 1% β-mercaptoethanol.
Autofluorescent erythrocytes were manually removed with the eraser tool.
Peel was manually removed with a ceramic knife, then the fruits were cored and the pulp was cut into 1 cm thick wedges.
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The vitteline membrane and all debris were manually removed and covered with a cover slip.
We then manually removed all PSGs with potential errors in their alignments to minimize the false discovery rate.
We then manually removed all candidate PSGs with potential errors in their alignments to minimize the false discovery rate.
The library was viewed under an Olympus SZX12 fluorescence microscope (Olympus America) (Supporting Figure s2), and red-colored beads were manually removed from the library with a micropipet.
After washing in Ca2+-free SOS to remove collagenase, the follicle cell layer was manually removed using forceps, and followed with the nuclear injection of 20 nl cDNAs of the chicken nAChR subunits (α3, α4, α7, β2 and β3) in the pcDNA3.1 expression vector in distilled water (final concentration of each cDNA: 0.1 ng nl−1).
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