2. There are many options for bead-based rRNA depletion, each
with differing protocols.
3. This computer should have enough hard drive capacity to store
the. FASTQ files that come from the sequencing run. Each
sequencing run will generate ~30 GB of raw data. When generating a large amount of sequencing data, the disk space
required will increase.
4. Both the .FASTA and the .GTF files can be obtained from
NCBI Genbank https://www.ncbi.nlm.nih.gov/assembly. To
ensure compatibility between the reference genome and annotations, we recommend that both the reference genome in
FASTA format and a file of gene annotations in. GTF format
are downloaded from the same assembly. The two files to
retrieve from the assembly are Genomic FASTA (.FNA) and
Genomic GTF (.GTF).
5. These steps are recommended to remove any trace amount of
beads but can be left out if desired.
6. To achieve sufficient RNA input quantities from dilute samples,
it is possible to concentrate samples by speed-vacuuming.
7. Before starting the Library preparation, it is best to start thermocycler and program and so the machine has reached the
correct lid temperature when you reach those steps.
8. Like rRNA depletion beads, there are many different options
for cDNA purification beads. Agencourt AMPure is the brand
of beads used in the following protocols and uses a solid phase
reversible immobilization (SPRI) technique.
9. During all bead-based steps, there is a 5 min drying period. If
working with multiple samples at a time, it is helpful to begin
counting the 5 min while mixing the first sample. This ensures
that you never exceed 5 min and reduce possible bead overdrying.
10. The adaptor is provided in NEBNext Singleplex (NEB
#E7350) or NEBNext Multiplex (NEB #E7335, #E7500,
#E6609, or #E7600) Oligos for Illumina.
11. A precipitate can form upon thawing of the NEBNext Hot
Start HiFi PCR Master Mix. To ensure optimal performance,
place the master mix at room temperature while purifying the
ligation reaction. Once thawed, gently mix by inverting the
tube several times.
12. Primers are provided in NEBNext Multiplex (NEB #E7335,
#E7500, and #E7600) Oligos for Illumina. For use with Dual
Index Primers (NEB #E7600), use only one i7 Primer and one
i5 Primer per reaction. For example, when using a 96-well
plate, the first row might be i71i51, i71i52, i71i53, etc. To
maximize usage of Multiplexing Primers across multiple
508
Shuyi Ma et al.
with differing protocols.
3. This computer should have enough hard drive capacity to store
the. FASTQ files that come from the sequencing run. Each
sequencing run will generate ~30 GB of raw data. When generating a large amount of sequencing data, the disk space
required will increase.
4. Both the .FASTA and the .GTF files can be obtained from
NCBI Genbank https://www.ncbi.nlm.nih.gov/assembly. To
ensure compatibility between the reference genome and annotations, we recommend that both the reference genome in
FASTA format and a file of gene annotations in. GTF format
are downloaded from the same assembly. The two files to
retrieve from the assembly are Genomic FASTA (.FNA) and
Genomic GTF (.GTF).
5. These steps are recommended to remove any trace amount of
beads but can be left out if desired.
6. To achieve sufficient RNA input quantities from dilute samples,
it is possible to concentrate samples by speed-vacuuming.
7. Before starting the Library preparation, it is best to start thermocycler and program and so the machine has reached the
correct lid temperature when you reach those steps.
8. Like rRNA depletion beads, there are many different options
for cDNA purification beads. Agencourt AMPure is the brand
of beads used in the following protocols and uses a solid phase
reversible immobilization (SPRI) technique.
9. During all bead-based steps, there is a 5 min drying period. If
working with multiple samples at a time, it is helpful to begin
counting the 5 min while mixing the first sample. This ensures
that you never exceed 5 min and reduce possible bead overdrying.
10. The adaptor is provided in NEBNext Singleplex (NEB
#E7350) or NEBNext Multiplex (NEB #E7335, #E7500,
#E6609, or #E7600) Oligos for Illumina.
11. A precipitate can form upon thawing of the NEBNext Hot
Start HiFi PCR Master Mix. To ensure optimal performance,
place the master mix at room temperature while purifying the
ligation reaction. Once thawed, gently mix by inverting the
tube several times.
12. Primers are provided in NEBNext Multiplex (NEB #E7335,
#E7500, and #E7600) Oligos for Illumina. For use with Dual
Index Primers (NEB #E7600), use only one i7 Primer and one
i5 Primer per reaction. For example, when using a 96-well
plate, the first row might be i71i51, i71i52, i71i53, etc. To
maximize usage of Multiplexing Primers across multiple
508
Shuyi Ma et al.