10. Ethidium bromide.
11. Thermocycler.
2.7 Selection
and Purification
of Library Fragments
1. Agarose.
2. 1Â TAE buffer.
3. Ethidium bromide.
4. Monarch DNA gel extraction kit (New England Biolabs).
5. Molecular-grade water.
2.8 Mapping Reads
1. Interface for using command line software (terminal on a Mac,
a UNIX workstation, or the command prompt application on
a PC).
2. Reference genome for your organism in fasta format.
3. Burrows-Wheeler Aligner (bwa, free download from
sourceforge.net) (or other mapping software).
4. Samtools (free download).
5. Bedtools (free download).
2.9 Removal of Read
2 and Separation
of Plus and Minus
Strands
1. Interface for using command line software (terminal on a Mac,
a UNIX workstation, or the command prompt application on
a PC).
2. Perl.
3. Python.
4. Scripts publicly available here: https://github.com/ssshell/5prime-end-mapping.
2.10 Determine Read
Depth for each 5
0 End
1. Interface for using command line software (terminal on a Mac,
a UNIX workstation, or the command prompt application on
a PC).
2. Bedtools.
3. Python.
4. Scripts publicly available here: https://github.com/ssshell/5prime-end-mapping.
2.11 Filter to Obtain
Above-Background 5
0
Ends and Ensure
Correct Normalization
Between Converted
and Nonconverted
Libraries
1. RNAseq expression data from the same strains/conditions
used for 5
0 end mapping.
2. Interface for using command line software (terminal on a Mac,
a UNIX workstation, or the command prompt application on
a PC).
3. Bedtools.
4. Python.
5. Scripts publicly available here: https://github.com/ssshell/5prime-end-mapping.
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