Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 71
3.2.4. Ligating Linkers to cDNA
1. After the NlaIII restriction digest, remove the supernatant and wash the beads
twice with 200 µL of 1X BW/0.1% SDS containing 2X BSA.
2. Wash four times with 200 µL of 1X BW containing 2X BSA (freshly made).
3. Wash twice with 200 µL of 1X ligase buffer.
4. Divide the beads into two tubes and place them on a magnet.
5. Remove the last wash and set up a 10-µL ligation with linker 1AB (1 µL) and
linker 2AB (1 µL) in separate tubes. The amount of linkers used needs to be
adjusted according to the number of cells (see Note 16). Two different linkers are
used to prevent looping of the template in the PCR reaction. The linkers encode
for the BsmfI restriction enzyme site.
6. Heat tubes at 50°C for 2 min and then let sit at room temperature for 15 min.
7. Add 1 µL of T4 high concentration ligase (5 U/µL) to each tube (10-µL reaction).
8. Incubate at 16°C for 2 h and mix beads intermittently by tapping the tube.
3.2.5. Release Tags by Using Tagging Enzyme (BsmfI) of cDNA
1. After ligation, wash each sample twice with 200 L of 1X BW/0.1% SDS containing
2X BSA (freshly made), by adding the 200 µL of buffer to the 10-µL
ligation reaction.
2. Pool tube 1 and tube 2 after the first wash to minimize losses in subsequent steps.
3. Wash four times with 200 µL of 1X BW/2X BSA (freshly made).
4. Wash twice with 200 µL of 1X NEB T4 with 2X BSA (freshly made).
5. Set up a restriction digest with BmsfI. BsmfI produces a 5′ sticky end of four bases
(10 bp + 4 bp into unknown sequence).
6. Incubate at 65°C for 1 h, mix intermittently, and then centrifuge at 14,000g for
2 min at 4°C.
7. Transfer supernatant to new tube (see Note 17).
8. Wash beads once with 40 µL of LoTE; thereafter, pool the 200-µL supernatant and
the 40-µL wash (240 µL final volume).
9. Perform phenol:chloroform extraction by adding 240 µL of phenol:chloroform,
pH 8.0 (“PC8”).
10. Vortex, then centrifuge for 5 min at 11,000g at 4°C and transfer the upper aqueous
phase to a new tube.
11. Then, precipitate with 100% ethanol (EtOH) at −80°C. Centrifuge for 30 min at
21,000g at 4°C. Wash twice with 200 µL of 75% EtOH.
12. Resuspend pellet in 10 µL of LoTE by pipeting up and down.
3.2.6. Blunt Ending Released Tags
The tags now have a protruding end that needs to be filled in for blunt end
ligation to form the ditags.
1. Set up blunt end ligation by using Klenow (2 U/µL).
2. Incubate at 37°C for 30 min.
3. Then, add 190 µL of LoTE (240 µL final volume).