2. Determine the cell density of each culture with the PetroffHausser chamber under a dark-field microscope (see Note 2).
3. Dilute each culture to the same cell density and pool equal
volumes together (input pool) (see Notes 3 and 4).
3.2 Fitness Testing
in Hamsters
All animal experiments must be approved by the local Institutional
Animal Care and Use Committee and must be performed under
biosafety level 2 (BSL2) containment.
Table 1
Oligonucleotide primers for semi-random PCR [10]
Oligonucleotide primers for semi-random PCR [10]
Name
Sequence (5
0 –3
0 )
Tnk1
CTTGTCATCGTCATCCTTG
Tnk2
GTGGCTTTATTGATCTTGGG
Deg1
a
GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT
Deg2
a
GGCCACGCGTCGACTAGTACNNNNNNNNNNTCTT
TnkN1
CGTCATGGTCTTTGTAGTCTATGG
TnKN2
TGGGGATCAAGCCTGATTGGG
Tag
GGCCACGCGTCGACTAGTAC
a
“NNNNNNNNNN” indicates a random nucleotide at each of the ten positions
Table 2
Oligonucleotide primers for Illumina sequencing [5, 6]
Oligonucleotide primers for Illumina sequencing [5, 6]
Name
Sequence (5
0 –3
0 )
TnkN3
CGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCC
TGATTGGG
olj376
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGGGGGGGGGGGGG
pMargent2 AATGATACGGCGACCACCGAGATCTACACTCTTTCCGGGGACTTA
TCAGCCAACCTGTTA
IP
a
CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTG
TGCTCTTCCGATCT
IP seq
GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
pMargent3 ACACTCTTTCCGGGGACTTATCAGCCAACCTGTTA
IP indexing primer
a
XXXXXX refers to a specific barcode that differs for each primer
100
Kristel Lourdault and James Matsunaga