Question: How do I generate custom panels from Twist Oligo Pools for use in the Targeted Gene Expression assay?
Answer: Custom panels can be generated from Twist Oligo Pools by following our Demonstrated Protocol CG000412. 10x Genomics officially supports the use of these panels as fully custom panels (human or mouse) in the Single Cell Targeted Gene Expression assay.
Prior to ordering the Twist Oligo Pool, primer binding sites must be appended to each bait sequence, as shown in Table 2 of CG000412. Follow the step-by-step instructions below to modify the bait sequences prior to ordering Twist Oligo Pools.
Instructions for modifying the bait design file for purchasing Twist Oligo Pools:
1) Download 10x Genomics bait design
After designing a custom panel, download the “Bait Design File” from the 10x Genomics Custom Panel Designer.
2) Copy bait names and sequences into a new spreadsheet
Copy and paste column C (Bait ID) into column A of a new Excel spreadsheet.
Copy and paste column B (Sequence) into column B of the new spreadsheet.
3) Trim baits
To reduce bait costs, it may be desirable to trim one nucleotide off both the 5’ and 3’ ends of baits that are 120 nucleotides (nt) in length. This will ensure that the final length of the baits plus primer binding sites is <= 150 nt.
This can be achieved by entering the following formula into column C:
4) Append primer binding sites
Select a set of 5’ and 3’ flanking sequences from Table 2 in Demonstrated Protocol CG000412. For this example, we will use the following flanking sequences:
5’ flanking sequence = TGAACGCGCTACTATG
3’ flanking sequence = ACTAACGTCGATTGGC
Append these sequences to the trimmed baits by entering the following formula into column D:
5) Create a new spreadsheet with modified bait sequences
Copy and paste column A (Bait ID) into column A of a new spreadsheet
Copy and paste column D (trimmed and appended bait sequences) into column B of a new spreadsheet. Use the "Paste Values" function to paste the final bait sequences rather than the formula. On a Mac computer, select “Edit” → “Paste Special” → “Values”. On a Windows computer, select "Home" → "Paste" → "Paste Values".
6) Use this spreadsheet to order Oligo Pools from Twist
Follow the instructions in Demonstrated Protocol CG000412 for ordering Twist Oligo Pools