Rehybridization of a HiSeq v4 high output flow cell is typically performed on the HiSeq instrument, but in some cases, a Read 1 rehybridization can be performed on the cBot. This bulletin describes the steps and reagents necessary to perform this rehybridization.

The following reagents and consumables are needed:

• FDR, SB2, and the Read 1 sequencing primer (HP10)** from a HiSeq Multi-Primer Rehyb v4 Kit v4 [GD-403-4001]
• Leftover HT1 (> 3.6 ml) from the HiSeq v4 cluster kit
• The HiSeq v4 high output sequencing flow cell to be rehybridized
• A used HiSeq v4 cBot cluster plate with the original tube strips*



*If a used HiSeq v4 clustering plate is not available, you can use 8-tube strips from a different cluster plate. The tube strips should be thoroughly cleaned with laboratory-grade water to avoid reagent contamination.

**If you are using custom primers, replace HP10 with 0.5 µM of your custom Read 1 sequencing primer.

Before starting, note the following:

• At least 3.6 ml of HT1 from the cluster kit is needed to perform the rehybridization
• The HiSeq v4 rehybridization recipes are available on v3.0.46 or later of the cBot software

Follow these steps to perform the rehybridization:

1. Thaw the SB2 in a room temperature water bath for 60 minutes.
2. Thaw the FDR and HP10 reagents in a room temperature water bath for 20 minutes.
3. Pipette the reagents into the tube strips in the following rows with the volumes indicated:
   • Row 1 – HT1 (at least 450 µl per tube)
   • Row 2 – HP10 (at least 150 µl per tube)
   • Row 5 – FDR (at least 250 µl per tube)
   • Row 7 – SB2 (at least 350 µl per tube)
4. Select the appropriate v4 rehybridization recipe based on the type of flow cell:
   • PE_HiSeq_Cluster_Kit_v4_cBot_Rehyb_recipe_v9.0
   • SR_HiSeq_Cluster_Kit_v4_cBot_Rehyb_recipe_v9.0
5. Load the flow cell and scan the used HiSeq v4 plate ID. If using a different type of reagent plate, manually input the reagent plate ID as GA1234567-PE4.
6. Load the cluster plate onto the cBot and follow the on-screen prompts to complete the run setup.

When the rehybridization is complete, use the rehybridized flow cell to set up a new run on the HiSeq.