Additional Indexes Guide - DNA, RNA, and Protein Libraries

If more than 8 samples need to be multiplexed on one lane on an Illumina sequencer, the following additional i5 and i7 indexes may be purchased from IDT and used for Library PCR by making unique pairs following the instructions below. Indexes are listed for DNA, RNA, and Protein libraries.

The following indexes can be used combinatorially to multiplex beyond 16 samples. For low-plex sequencing using these indexes, select combinations that give diversity as per Illumina's best practices.

Order

1. Order oligos (single-stranded DNA) on IDT: https://www.idtdna.com/pages/support/how-to-order
2. Purification method:
   • DNA: standard desalting, 25 nmole
   • RNA: i5 index primers standard desalting, 25 nmole; i7 index primers standard desalting, 4 nmole Ultramer DNA oligo
   • Protein: standard desalting, 4 nmole Ultramer DNA oligo

Library PCR Setup

3. Dilute each oligo to 4 µM with nuclease-free water (typically from 100 µM stock)
4. For each sample combine 5 µl of i5 index primer and 5 µl of i7 index primer. The combined primers represent the "DNA Index Primer", "RNA Index Primer", or "Protein Index Primer"
5. Proceed as outlined in the User Guide

The index sequence within each primer is shown in bold. The i7 Index Sequence column lists the reverse complement of the bolded sequence, as entered in the sample sheet.

DNA i5 Index Primers

DNA i7 Index Primers

RNA i5 Index Primers

RNA i7 Index Primers

Protein i5 Index Primers

Protein i5 index primers are identical to the DNA i5 index primers.

Protein i7 Index Primers

• Additional Indexes DNA RNA Protein.xlsx9 KB