6. Divide into as many microcentrifuge tubes as necessary and add
approximately 3 volumes Dissolving Buffer. The number of
tubes and volume of Dissolving Buffer will depend on the
size of the gel slice, which will vary depending on the fragmentation conditions as well as the width of the comb and how
long the gel was run.
7. Melt gel slices at room temperature, vortexing periodically.
Allow approximately 2 h for this step.
8. Apply 700 μL of melted gel in Dissolving Buffer to Gel Extraction column, centrifuge for 1 min at >15,000 Â g, and discard
flow-through.
9. Repeat step 8 as needed with additional melted gel.
10. Apply 400 μL of Wash Buffer to column, centrifuge for 1 min
at >15,000 Â g, and discard flow-through.
11. Repeat step 11 for a total of two washes.
12. Centrifuge for 1 min at >15,000 Â g to ensure membrane is
dry and then transfer column to a clean 1.5 mL tube.
13. Pipet 20 μL of molecular-grade water onto membrane, centrifuge for 1 min at >15,000 Â g, and leave flow-through in tube.
14. Repeat step 14 for a total elution volume of 40 μL.
15. Use a spectrophotometer to determine the 260/230 ratio of
product. If the ratio is <1, perform the KAPA bead-based
purification described in Subheading 3.5. If the ratio is >1,
go directly to sequencing.
16. Libraries can now be run on any Illumina sequencer (HiSeq,
Genome Analyzer, or MiSeq) using a paired-end protocol and
sequencing primers appropriate for TruSeq genomic DNA
libraries.
3.8 Alignment
to a Reference Genome
1. Obtain demultiplexed fastq files from your Illumina instrument
or sequencing facility.
2. Obtain a reference genome in fasta format from NCBI or
another source (see Note 13). On the command line, use the
bwa mem command to align read 1 and read 2 of each library to
the reference genome, producing sam files (see Fig. 2 and
Note 14).
3. On the command line, use the samtools view command to
produce .bam files from the sam files.
4. On the command line, use the samtools sort command to sort
each .bam file.
3.9 Removal of Read
2 and Separation
of Plus and Minus
Strands
1. As a result of the library construction method, read 1 represents
the sequence of an endogenous RNA, with the first nt of the
read representing the 5
0 most nt of that RNA molecule. Read
2 represents the reverse complement of part of an endogenous
524
M. Carla Martini et al.
approximately 3 volumes Dissolving Buffer. The number of
tubes and volume of Dissolving Buffer will depend on the
size of the gel slice, which will vary depending on the fragmentation conditions as well as the width of the comb and how
long the gel was run.
7. Melt gel slices at room temperature, vortexing periodically.
Allow approximately 2 h for this step.
8. Apply 700 μL of melted gel in Dissolving Buffer to Gel Extraction column, centrifuge for 1 min at >15,000 Â g, and discard
flow-through.
9. Repeat step 8 as needed with additional melted gel.
10. Apply 400 μL of Wash Buffer to column, centrifuge for 1 min
at >15,000 Â g, and discard flow-through.
11. Repeat step 11 for a total of two washes.
12. Centrifuge for 1 min at >15,000 Â g to ensure membrane is
dry and then transfer column to a clean 1.5 mL tube.
13. Pipet 20 μL of molecular-grade water onto membrane, centrifuge for 1 min at >15,000 Â g, and leave flow-through in tube.
14. Repeat step 14 for a total elution volume of 40 μL.
15. Use a spectrophotometer to determine the 260/230 ratio of
product. If the ratio is <1, perform the KAPA bead-based
purification described in Subheading 3.5. If the ratio is >1,
go directly to sequencing.
16. Libraries can now be run on any Illumina sequencer (HiSeq,
Genome Analyzer, or MiSeq) using a paired-end protocol and
sequencing primers appropriate for TruSeq genomic DNA
libraries.
3.8 Alignment
to a Reference Genome
1. Obtain demultiplexed fastq files from your Illumina instrument
or sequencing facility.
2. Obtain a reference genome in fasta format from NCBI or
another source (see Note 13). On the command line, use the
bwa mem command to align read 1 and read 2 of each library to
the reference genome, producing sam files (see Fig. 2 and
Note 14).
3. On the command line, use the samtools view command to
produce .bam files from the sam files.
4. On the command line, use the samtools sort command to sort
each .bam file.
3.9 Removal of Read
2 and Separation
of Plus and Minus
Strands
1. As a result of the library construction method, read 1 represents
the sequence of an endogenous RNA, with the first nt of the
read representing the 5
0 most nt of that RNA molecule. Read
2 represents the reverse complement of part of an endogenous
524
M. Carla Martini et al.