babraham.ac.uk/projects/fastqc/), ShortStack version !3.8.5
[53, 54], Bowtie version !1.2.2 [55] and SAMtools version
!1.9 [56], JBrowse [57]/Integrative Genomics Viewer (version !2.7.0) [58, 59], and Geneious Prime v2019.0 (https://
www.geneious.com/).
2.3 RNA Extraction,
Size Fractionation, and
Northern Blotting
1. Autoclaved, pre-cooled ceramic mortars and pestles, and a
metal spatula.
2. Liquid nitrogen.
3. 13 mL Round-bottomed, capped polypropylene tubes.
4. Beckman centrifuge, rotor JA17 or JA-25.5, and fitted rubber
adapters.
5. TRIzol (Invitrogen, CA, USA): Store at 4
C (see Note 1).
6. Serological 10 mL pipettes (single-use) and an electric pipette
controller.
7. Cold chloroform: Store at À20
C (see Note 1).
8. Cold isopropanol: Store at À20
C.
9. Absolute ethanol: Store at RT.
10. 75% Ethanol (in Milli-Q water): Store at À20
C.
11. Diethyl pyrocarbonate (DEPC)-treated water: DEPC inactivates RNases in aqueous solution. Prepare a 0.1% DEPC solution—e.g., 1 mL of DEPC diluted in 1 L Milli-Q water. Leave
this solution at room temperature (RT) for 4 h and then
autoclave (>15 min) to remove the DEPC. Store at RT (see
Note 1).
12. RNase-free 1.5 mL Eppendorf tubes (also referred to as microfuge tubes).
13. NanoDrop-2000 Spectrophotometer (Thermo Fisher Scientific, MA, USA).
14. RNeasy Midi Kit (QIAGEN) or RNeasy Mini Kit, containing
the RLT and RPE buffers.
15. β-Mercaptoethanol: Store at RT (see Note 1).
16. RNase-free 30 mL Corex glass centrifuge tubes.
17. A medium-sized polyacrylamide gel electrophoresis (PAGE)
system (see Note 2).
18. 5Â TBE stock solution: 54 g Tris base, 27.5 g boric acid,
20 mL 0.5 M ethylenediaminetetraacetic acid (EDTA,
pH 8.0) filled up with Milli-Q water to 1 L. Store at RT.
19. 50 mL Falcon tubes.
20. Acrylamide/bis-acrylamide (19:1): Store at 4
C (see Note 1).
Genome-Scale and Northern Blot Analyses of siRNAs
393
[53, 54], Bowtie version !1.2.2 [55] and SAMtools version
!1.9 [56], JBrowse [57]/Integrative Genomics Viewer (version !2.7.0) [58, 59], and Geneious Prime v2019.0 (https://
www.geneious.com/).
2.3 RNA Extraction,
Size Fractionation, and
Northern Blotting
1. Autoclaved, pre-cooled ceramic mortars and pestles, and a
metal spatula.
2. Liquid nitrogen.
3. 13 mL Round-bottomed, capped polypropylene tubes.
4. Beckman centrifuge, rotor JA17 or JA-25.5, and fitted rubber
adapters.
5. TRIzol (Invitrogen, CA, USA): Store at 4
C (see Note 1).
6. Serological 10 mL pipettes (single-use) and an electric pipette
controller.
7. Cold chloroform: Store at À20
C (see Note 1).
8. Cold isopropanol: Store at À20
C.
9. Absolute ethanol: Store at RT.
10. 75% Ethanol (in Milli-Q water): Store at À20
C.
11. Diethyl pyrocarbonate (DEPC)-treated water: DEPC inactivates RNases in aqueous solution. Prepare a 0.1% DEPC solution—e.g., 1 mL of DEPC diluted in 1 L Milli-Q water. Leave
this solution at room temperature (RT) for 4 h and then
autoclave (>15 min) to remove the DEPC. Store at RT (see
Note 1).
12. RNase-free 1.5 mL Eppendorf tubes (also referred to as microfuge tubes).
13. NanoDrop-2000 Spectrophotometer (Thermo Fisher Scientific, MA, USA).
14. RNeasy Midi Kit (QIAGEN) or RNeasy Mini Kit, containing
the RLT and RPE buffers.
15. β-Mercaptoethanol: Store at RT (see Note 1).
16. RNase-free 30 mL Corex glass centrifuge tubes.
17. A medium-sized polyacrylamide gel electrophoresis (PAGE)
system (see Note 2).
18. 5Â TBE stock solution: 54 g Tris base, 27.5 g boric acid,
20 mL 0.5 M ethylenediaminetetraacetic acid (EDTA,
pH 8.0) filled up with Milli-Q water to 1 L. Store at RT.
19. 50 mL Falcon tubes.
20. Acrylamide/bis-acrylamide (19:1): Store at 4
C (see Note 1).
Genome-Scale and Northern Blot Analyses of siRNAs
393