title: Huperzia selago raw sequence reads
design: Preparation and storage of the shoot apices for RNA isolation  Leaves were removed from the shoots with a razor. Shoot tips were divided into several samples of 100 mg FW each. The samples were ground with mortar and pestle upon addition of 100 mg Polyclar AT (Serva, Mannheim, Germany) in liquid nitrogen. The ground samples were transferred into Eppendorf tubes pre-cooled in liquid nitrogen and stored at -80°C until being used for RNA isolation.  Total RNA extraction protocol  RNA was isolated using TRIzol reagent (Chomczynski P, Mackey K, 1995). For preparation of 10 ml TRIzol reagent, 3.8 ml liquid phenol, 2 ml guanidine thiocyanate (4 M), 1 ml ammonia thiocyanate (4 M), 334 µl Na acetate (3 M, pH 5.0) and 500 µl 100% glycerol were mixed and the volume was adjusted to 10 ml with distilled water. RNA was isolated using the following protocol:  • add 1 ml of TRIZol reagent to a frozen sample of H. selago shoot apices (see above). The sample is thawn by vortexing and incubated for 5 min at RT with occasional vortexing  • 10 min centrifugation at maximal rpm at 4°C  • add 200 µl chloroform to the supernatant; mix for 15 s by hand, incubate for  2-3 min at RT  • 15 min centrifugation at 13 krpm at 4°C  • add ½ volume of isopropanol and ½ volume of the solution containing Na citrate (0.8 M) and NaCl (1.2 M) to the supernatant  • mix by inverting the Eppendorf tube several times, incubate for 10 min at RT  • 10 min centrifugation at 12,000 x g at 4°C.  • Discard supernatant; add 300 µl of Na acetate (3 M, pH 5.2) and resuspend the pellet  • 10 min centrifugation at 12,000 x g at 4°C  • Wash the sediment with 75 % EthOH twice (add 900 µl of 75 % EtOH, centrifuge at 7500 x g for 5 min at 4°C, discard supernatant, repeat)  • dry the pellet in open Eppendorf tube at 37°C for 5 min, re-dissolve in 30 µl of distilled sterile Н2О by warming the tube for 15 min at 65°C  All solutions were RNAse-free. The isolated RNA was stored at -80°С.  Numbers of samples used for the extractions and contamination analyses  Altogether, 8 samples of 100 mg each were used (FIGURE 1). Total RNA isolated from samples № 2, 5, 7, 8 were combined into one sample (the tube was marked “Huperzia selago total RNA №1”, total volume 100 µl). In these samples, no contamination with genomic DNA could be seen on gel. The presence of the DNA contamination was further checked with ubiquitin-PCR:   94°C 3 min  94°C 30 s  54°C 30 s  72°C 1.5 min     30 cycles  72°C 5 min  12°C forever  The PCR revealed no contamination. Therefore, the RNA in this tube was not subjected to DNase treatment. The RNA was precipitated by addition of 10 µl Na acetate (3 M, pH 5.2) and 102.5 µl of 96 % EthOH. After addition, the tube was stored at  -80°C until being transported on ice to Stockholm.