A Practical Guide to Cancer Systems Biology

18 A Practical Guide to Cancer Systems Biology

• Incubate the sample at room temperature for 30 seconds, then remove
  and discard all of the supernatant from each well.


• Repeat the 80% EtOH wash steps twice.


• Open the lid and let the sample tube stand at room temperature for 15
  minutes to dry and then remove the sample from the magnetic stand.


• Add 17.5μL RSB to each sample. Gently pipette the entire volume up
  and down 10 times to mix thoroughly.


• Incubate the sample at room temperature for 2 minutes.


• Centrifuge briefly and place the sample on the magnetic stand at room
  temperature for 5 minutes.


• Transfer 15μL of the clear supernatant to a new PCR tube.

Note: The purified ds DNA can be stored at−15 –− 25 ◦Cforupto7days.

VI.Adenylate 3′ends

• Pre-heat the thermal cycler to 37◦C.


• Prepare the in-line control reagent by diluting the CTA to 1/100 in RSB
  (1μLCTA+99μL RSB) before use. Add 2.5μL of dilute CTA to each
  sample that contains 15μL of ds DNA. If not using the in-line control
  reagent, add 2.5μL of RSB instead.


• Add 12.5μL of ATL to each sample. Gently pipette the entire volume up
  and down 10 times to mix thoroughly.


• Place the sample on the thermal cycler to pre-form end repair.

37 ◦C for 30 minutes

• Remove the sample from the thermal cycler, centrifuge briefly, and proceed
  immediately to Adapter Ligation.

VII.Ligate adapters

• Pre-heat the thermal cycler to 30◦C.


• Prepare the in-line control reagent by diluting the CTL to 1/100 in RSB
  (1μL CTL + 99μL RSB) before use. Add 2.5μL of dilute CTL to each
  sample. If not using the in-line control reagent, add 2.5μL of RSB instead.


• Before use, immediately remove the Ligation Mix tube from− 15 ◦Cto
  − 25 ◦C storage. Add 2.5μL of Ligation Mix to each sample. Gently pipette
  the entire volume up and down 10 times to mix thoroughly.


• Return the Ligation Mix tube back to− 15 ◦Cto− 25 ◦C storage immedi-
  ately after use.


• Add 2.5μL of appropriate/desired thawed RNA Adapter Index to each
  sample. Gently pipette the entire volume up and down 10 times to mix
  thoroughly.