STICR Barcode Library Amplification Protocol

Following cDNA amplification in 10X workflow, bead purify as directed in instructions and set aside 10ul of cDNA to use in the following reaction. This should leave you with 30µL of bead purified cDNA to complete the rest of the standard 10X whole transcriptome library with.

Primers: Reverse primer (P5-Read1) plus a forward primer (P7-i7 index-Read2-Upstream_Barcode sequence). If you plan to sequence multiple barcode libraries on the same lane, you will need to use different reverse primers as they will need to have different i7 indexes.

PCR Reaction Mix

Amplify library with standard NEB Protocol for Q5 Hot Start High-Fidelity 2X Master Mix in 50µL reaction:

1. 25µLQ5 High-fidelity 2X Master Mix

2. 2.5µL 10micromolar (µM) i7_indexed Reverse Primer (283-290)

3. 2.5µL 10micromolar (µM)i5_indexed Forward Primer (291-298)

4. 10µL molecular grade H₂0

5. 10µL 10X cDNA

PCR program:

1. 98°C for 0h 0m 30sseconds
2. 98°C for 0h 0m 10sseconds
3. 62°C for0h 0m 20sseconds
4. 72°C for 0h 0m 10sseconds
5. Repeat steps 2 through 4 ~15X (if unsure, run tests with 2uL starting cDNA at a range of cycles, but do not exceed 20 cycles for final preparation)
6. 72°C for 0h 2m 0sminutes
7. 4°CHold

Perform dual-sided SPRI selection

Add 30µL of SPRI beads to 50µL of PCR reaction, mix by pipetting 15 times.

Incubate at RT for 0h 5m 0sminutes

Place on 10X magnet on High for 0h 3m 0sminutes. DO NOT discard supernatant.

Transfer supernatant to new PCR tube.

Add 10µL of SPRI beads, mix by pipetting 15 times.

Incubate at RT for 0h 5m 0sminutes

Place on 10X magnet on High for 0h 3m 0sminutes

Carefully remove and discard supernatant (do not disrupt beads).

Wash beads with 200µL of freshly prepared 80% EtOH.

Let stand 0h 0m 30sseconds, remove EtOH.

Wash 1 additional time with 80% EtOH for 0h 0m 30sseconds

Remove EtOH with pipette, briefly centrifuge and return PCR tube to 10X magnet in low position. Remove any residual EtOH with pipette.

Add 22µL of Buffer EB, mix by pipetting 15 times.

Let stand 0h 5m 0sminutes RT.

Place on 10X magnet on low for 0h 3m 0sminutes

Remove supernatant to new PCR tube. This is your barcode library.

You can confirm library prep with Agilent BioA/Tapestation.Trace should look like:

Ensure that separate libraries were prepared with different antibodies before pooling and submit for sequencing ~30 million reads per library.