Synthesis EecSeq

---

• Place the plate/tubes in a thermal cycler and carry out the fragmentation and priming program as follows:

Desired Fragment Size	Temp.	Duration
100 – 200 bp	94 °C	8 min
200 – 300 bp	94 °C	6 min
300 – 400 bp	85 °C	6 min

For Puritz and Lotterhos (2017), we chose 94 °C for 7 mins to have fragments between 150-250 bp, approximately the same size distribution as planned for our DNA libraries.

• Immediately place the plate/tube on a magnet to capture the beads, and incubate until the liquid is clear. Caution: To prevent hybridization of poly(A)- rich RNA to the capture beads, do not allow the sample to cool before placing on the magnet-
• Carefully remove 10 μl of the supernatant containing the eluted, fragmented, and primed RNA into a separate plate or tube.
• Proceed immediately to 1st Strand Synthesis.

1st Strand Synthesis

• On ice, assemble the 1st Strand Synthesis reaction as follows:

Component	Volume
Fragmented, primed RNA eluted from beads	10 μl
1st Strand Synthesis Master Mix	5 μl
–Total Volume–	–15 μl–

• Keeping the plate/tube on ice, mix thoroughly by gently pipetting the reaction up and down several times.
• Incubate the plate/tube using the following protocol:

Step	Temp.	Duration
Primer extension	25 °C	10 min
1st Strand synthesis	42 °C	15 min
Enzyme inactivation	70 °C	15 min
HOLD	4 °C	∞

• Place the plate/tube on ice and proceed immediately to –2nd Strand Synthesis and Marking–.

2nd Strand Synthesis and Marking

• Assemble the 2nd Strand Synthesis and Marking reaction as follows:

Component	Volume
1st Strand cDNA	15 μl
2nd Strand Synthesis and Marking Master Mix	15 μl
–Total Volume–	–30 μl–

• Mix thoroughly by gently pipetting the reaction up and down several times.
• Incubate the plate/tube using the following protocol:

Step	Temp.	Duration
2nd Strand synthesis and marking	16 °C	60 min
HOLD	4 °C	∞

• Place the plate/tube on ice and proceed immediately to –2nd Strand Synthesis and Marking Cleanup–.