21. N,N,N
0 ,N
0 -tetramethylethylenediamine (TEMED): Store at
4
C.
22. 10% Ammonium persulfate (APS) in Milli-Q water: Aliquots
can be stored at À20
C for several months.
23. 10 mL Syringes with a 0.8 Â 40 mm needle.
24. DNA oligonucleotides used for PCR amplification prior to
Klenow probe synthesis, or for individual 5
0 -end-labeled
probes (sequences of the used DNA oligonucleotides listed in
Subheading 2.4).
25. RNA size standards for PAGE: Mix synthetic RNA oligonucleotides of 15 nt (2.5 μL), 21 nt (1.5 μL), 24 nt (1 μL), 30 nt
(0.5 μL), and 40 nt (0.5 μL) length (100 μM stocks; sequences
listed in Subheading 2.5). These RNA oligos should not be
homologous to the genome of interest. Add 2 μl low-range
ssRNA ladder (e.g., New England Biolabs, MA, USA) and
bring this mixture to the same total volume as the RNA sample
aliquots.
26. DynaMarker Prestain Marker “small RNA Plus” (BioDynamics
Laboratory Inc., Tokyo, Japan) used to follow small RNA
migration during gel electrophoresis.
27. SpeedVac Concentrator Savant (Thermo Fisher Scientific).
28. RNA-loading buffer: 95% Formamide, 0.025% bromophenol
blue, 0.025% xylene cyanol FF, 5 mM EDTA, 0.025% SDS,
pH 8.5.
29. High-current power supply for gel migration and transfer, such
as PowerPac HC (Bio-Rad, CA, USA).
30. Ethidium bromide (EtBr, 10 mg/mL) solution: Store at 4
C
and protected from light.
31. Digital gel documentation system with UV transillumination.
32. Positively charged nylon membrane (Hybond-N+).
33. Whatman paper.
34. A rolling device (e.g., a plastic serological pipette).
35. A magnetic stirrer.
36. Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) for RNA
electroblotting.
37. UV crosslinker to fix RNA to nylon membrane.
38. 2Â Saline sodium citrate (SSC): 0.3 M NaCl, 30 mM sodium
citrate, pH 7.0.
39. PerfectHyb Plus Hybridization Buffer (Sigma-Aldrich).
40. Hybridization equipment: Glass hybridization tubes, an oven
for the hybridizations, etc.
394
Marcel Bo ¨ hrer et al.
0 ,N
0 -tetramethylethylenediamine (TEMED): Store at
4
C.
22. 10% Ammonium persulfate (APS) in Milli-Q water: Aliquots
can be stored at À20
C for several months.
23. 10 mL Syringes with a 0.8 Â 40 mm needle.
24. DNA oligonucleotides used for PCR amplification prior to
Klenow probe synthesis, or for individual 5
0 -end-labeled
probes (sequences of the used DNA oligonucleotides listed in
Subheading 2.4).
25. RNA size standards for PAGE: Mix synthetic RNA oligonucleotides of 15 nt (2.5 μL), 21 nt (1.5 μL), 24 nt (1 μL), 30 nt
(0.5 μL), and 40 nt (0.5 μL) length (100 μM stocks; sequences
listed in Subheading 2.5). These RNA oligos should not be
homologous to the genome of interest. Add 2 μl low-range
ssRNA ladder (e.g., New England Biolabs, MA, USA) and
bring this mixture to the same total volume as the RNA sample
aliquots.
26. DynaMarker Prestain Marker “small RNA Plus” (BioDynamics
Laboratory Inc., Tokyo, Japan) used to follow small RNA
migration during gel electrophoresis.
27. SpeedVac Concentrator Savant (Thermo Fisher Scientific).
28. RNA-loading buffer: 95% Formamide, 0.025% bromophenol
blue, 0.025% xylene cyanol FF, 5 mM EDTA, 0.025% SDS,
pH 8.5.
29. High-current power supply for gel migration and transfer, such
as PowerPac HC (Bio-Rad, CA, USA).
30. Ethidium bromide (EtBr, 10 mg/mL) solution: Store at 4
C
and protected from light.
31. Digital gel documentation system with UV transillumination.
32. Positively charged nylon membrane (Hybond-N+).
33. Whatman paper.
34. A rolling device (e.g., a plastic serological pipette).
35. A magnetic stirrer.
36. Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) for RNA
electroblotting.
37. UV crosslinker to fix RNA to nylon membrane.
38. 2Â Saline sodium citrate (SSC): 0.3 M NaCl, 30 mM sodium
citrate, pH 7.0.
39. PerfectHyb Plus Hybridization Buffer (Sigma-Aldrich).
40. Hybridization equipment: Glass hybridization tubes, an oven
for the hybridizations, etc.
394
Marcel Bo ¨ hrer et al.