Prepare Reagents
Before setting up the run, prepare all reagents for sequencing: SBS reagents, indexing reagents, and paired-
end reagents, if applicable. Load all reagents when prompted by the control software. When using HiSeq v4
chemistry, returning to the instrument during the run to load reagents is not necessary.
Reagents can be prepared during cluster generation. For instructions on preparing clustering reagents and
performing cluster generation, see the
cBot System Guide (document # 15006165)
.
Split Reagents
It is possible to split a 250-cycle kit into 2 sets of reagents to perform shorter runs. For more information, see
.
Prepare SBS Reagents
SBS reagents are loaded onto the instrument at the beginning of the run. To prepare reagents, use the
following instructions to thaw SBS reagents.
Thaw SBS Reagents
1
Remove CRM, IRM, and USM from -25°C to -15°C storage. Protect IRM from light.
2
Thaw at 2°C to 8°C for about 16 hours.
Alternatively, thaw IRM and USM in a room temperature deionized water bath for about 90 minutes. Thaw
CRM in a
separate
water bath.
NOTE
Always replace your gloves after handling CRM.
3
Invert each bottle to mix.
4
Set IRM and USM aside on ice. Set CRM aside on ice
separately
to prevent cross-contamination.
5
Use PW1, SB1, SB2, and SB3 directly from storage.
Prepare Indexing Reagents
Indexing reagents are used during the indexing reads of an indexed sequencing run.
The PE and SR cluster kits include HP12, which is used for Index 1 (i7) Read 1, regardless of flow cell type.
Only the SR cluster kit includes HP9, which is required for a dual-indexed run on a single-read flow cell.
Run Type
Flow Cell Type
Index 1 (i7)
Index 2 (i5)
Single-indexed paired-end
PE
HP12
--
Single-indexed single-read
SR
HP12
--
Dual-indexed paired-end*
PE
HP12
--
Dual-indexed single-read
SR
HP12
HP9
* Dual-indexed paired-end runs use FRM, a paired-end reagent, for Index 2.
Thaw Indexing Reagents
1
Remove the following reagents from -25°C to -15°C storage:
u
For all indexed runs on a paired-end flow cell—FDR and HP12
Document # 15011190 v03
For Research Use Only. Not for use in diagnostic procedures.
12
HiSeq 2000 System Guide