2.10 Pme I Digestion
1. Gel electrophoresis casts, trays, combs, tank, and power device.
2. Incubator.
3. Nano drop.
4. 1.5 mL Eppendorf tubes and all size tips.
5. Autoclaved double-distilled water.
6. Blades.
7. Kim wipe.
8. QiaQuick gel extraction kit.
9. Pme I (together with buffer).
10. 0.5Â TBE buffer.
11. Ethidium bromide (EB).
12. 1 kb DNA ladder.
13. Low-melting agarose and regular agarose.
14. 10 mM Tris–HCl pH 8.0.
2.11 Computing
Analysis Workflow
Software listed in Table 3.
3 Methods
3.1 Synthesis of the
5
0 Pre-adenylated 3
0
Adapter
Oligonucleotides
This section is to provide a step-by-step protocol to synthesize a
large amount of 5
0 pre-adenylated 3 adapters for the 3
0 end ligation
of sRNAs. The protocol is developed from an early published
method [11] with numerous modifications for simplification and
friendly usage for a common laboratory.
Table 3
Software used in bioinformatic analysis
Software
Source
Website
Bowtie v1.1.2
[31]
http://bowtie-bio.sourceforge.net/index.shtml
FASTX-Toolkit
v0.0.13
The Hannon
Lab
http://hannonlab.cshl.edu/fastx_toolkit/
Samtools
N/A
http://samtools.sourceforge.net/
HTSeq v0.11.1
[35]
https://htseq.readthedocs.io/en/release_0.11.1/
SARTools
[37]
https://github.com/PF2-pasteur-fr/SARTools
FastQC v0.11.8
N/A
http://www.bioinformatics.babraham.ac.uk/projects/
fastqc/
234
Di Sun et al.
1. Gel electrophoresis casts, trays, combs, tank, and power device.
2. Incubator.
3. Nano drop.
4. 1.5 mL Eppendorf tubes and all size tips.
5. Autoclaved double-distilled water.
6. Blades.
7. Kim wipe.
8. QiaQuick gel extraction kit.
9. Pme I (together with buffer).
10. 0.5Â TBE buffer.
11. Ethidium bromide (EB).
12. 1 kb DNA ladder.
13. Low-melting agarose and regular agarose.
14. 10 mM Tris–HCl pH 8.0.
2.11 Computing
Analysis Workflow
Software listed in Table 3.
3 Methods
3.1 Synthesis of the
5
0 Pre-adenylated 3
0
Adapter
Oligonucleotides
This section is to provide a step-by-step protocol to synthesize a
large amount of 5
0 pre-adenylated 3 adapters for the 3
0 end ligation
of sRNAs. The protocol is developed from an early published
method [11] with numerous modifications for simplification and
friendly usage for a common laboratory.
Table 3
Software used in bioinformatic analysis
Software
Source
Website
Bowtie v1.1.2
[31]
http://bowtie-bio.sourceforge.net/index.shtml
FASTX-Toolkit
v0.0.13
The Hannon
Lab
http://hannonlab.cshl.edu/fastx_toolkit/
Samtools
N/A
http://samtools.sourceforge.net/
HTSeq v0.11.1
[35]
https://htseq.readthedocs.io/en/release_0.11.1/
SARTools
[37]
https://github.com/PF2-pasteur-fr/SARTools
FastQC v0.11.8
N/A
http://www.bioinformatics.babraham.ac.uk/projects/
fastqc/
234
Di Sun et al.