The 10x Chromium 3’ Reagent Kit was developed for amplification of 3’-ends of polyA+ RNAs and based on ligation of an adaptor upstream of cDNA at a variable distance from the poly-A+ priming site. The ligated adaptor is used as a binding site for a primer to read into the mRNA coding sequence. The sequence read from this upstream adaptor primer is typically only just 100 bases or so. The CloneTracker XP BC14-spacer-BC30 barcode is located at a fixed ~150-200nt distance upstream from the poly-A+ site, depending on the particular vector.
To effectively read the barcode sequence in the CloneTracker XP Barcode-3’ Library when using the 10x Genomics Chromium 3’ Kit, you need to include an additional PCR amplification step with a barcode-specific primer that is located just upstream of the CloneTracker XP barcode. This will ensure amplification of the segment of cDNA that contains both the CloneTracker XP barcode and the cell barcode associated with the poly-A+ sequence in the scRNA-Seq protocol (e.g., the “bead-barcode” with bead-based scRNA systems). The general procedure for how to integrate this additional PCR with the 10x Chromium 3’ Kit (Version 3.1) procedure is shown below.
Please note that the procedure below describes barcode/sgRNA detection when using the Chromium 3’-Kits from 10x Genomics. Depending on the specific scRNA-Seq protocol you are using, some modifications and optimization will be required.
Amplification of Barcodes and/or sgRNA for NGS when Using 10x Single Cell Analysis.
After the cDNA amplification/cleaning/quantitation step (Step 2.4 in Chromium Next GEM Single Cell 3ʹ v3.1, User Guide, Rev E), retrieve a ~10 ul (~25%) aliquot of 10x cDNA and set up a separate PCR reaction just before the cDNA fragmentation step.
- Amplify the barcodes/sgRNA by running a PCR reaction with the same cycling conditions as the previous cDNA amplification (Section 2.2 of the Chromium Next GEM Manual) and the following PCR primer below for your library:
(A) The Partial Read 1 primer (5’-CTACACGACGCTCTTCCGATCT-3’ — see 10x Chromium 3’ Kit User Guide)
for one PCR primer,
and
(B) 1 uM of either of the following primers for the second PCR primer (depending on the library you are using):
Adaptor-FBP1 primer — For CloneTracker XP Barcode-3’ Libraries in pScribe vectors:
Adaptor (Read 2) FBP1 primer
5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCGACCACCGAACGCAACGCACGCA-3’
Adaptor-U6 primer — For sgRNA libraries in pRSGScribe1 and pRSGScribe1cb:
Adaptor (Read 2) U6 primer
5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC-3’
- The standard Illumina adaptor sequence is located in the first 34 nucleotides of the 5’ end (underlined).
* Note: scRNA-Seq platforms other than 10x Chromium may use a different adaptor, in which case you should modify the primer accordingly.
* Note: The above PCR takes the place of the adaptor ligation step in the standard 10x protocol. Do not perform the Fragmentation, Adaptor Ligation, or post-Ligation SPRI Bead clean up for the amplified barcodes. Jump directly to the Sample Index PCR (Section 3.5 of the Chromium Next GEM Manual) and amplify the adaptor-ligated cDNA and barcodes using different i7 and i5 index primers as described in Step 2 below.
- Amplify the barcode and adaptor-ligated cDNA that was made in Step 3.3 of the 10x of the Chromium Next GEM Manual. Use 5 µl of the Barcode amplification mix from Step 1 above as the template to set up the Index Primer PCR using the of the reagents from 10x kit and the program described in Step 3.5 of the 10x Chromium Next GEM Manual.
Use a different set of i5 and i7 index primers for the Barcode Sample than used for the cDNA sample and, also, add an additional 6 PCR cycles for the barcode sample. The 6 additional PCR cycles will increase the yield of the CloneTracker XP barcoded cDNA sequences to approximately 64-fold higher levels than other transcripts in the cDNA reaction. This helps ensure you recover the majority of the CloneTracker XP barcodes.
- Run a Bioanalyzer analysis after amplification. You should be able to see a band of ~350-375nt (depending on the particular library), which corresponds to the barcode product amplified from lentiviral barcoded transcript.
The amplified product will have the following structure—Read2(Adaptor)-FBP1-Barcode-UMI-10xBarcode-Read1—which is the same structure as PCR products generated from amplified adaptor-ligated cDNA, except it has the CloneTracker barcode sequence instead of the cDNA—Read2(Adaptor)-cDNA-UMI-10xBarcode-Read1. If you can’t see the barcode-specific product, add an additional 2-4 extra cycles and repeat Bioanalyzer analysis. Then, quantitate them, purify using SPRI beads, and mix at the desired ratios for the follow-up NGS step.
- Purify the Indexed Barcode Amplicon as described in Post Sample Index PCR Double Sided Size Selection (Section 3.6 of the Chromium Next GEM Manual) of the 10x protocol. However, increase the amount of the SPRIselect Reagent added in Step e from 20 µl to 80 µl (1.4x). In other words, change Step e to the following:
e. Vortex to resuspend the SPRIselect reagent. Add 80 µl SPRIselect Reagent (1.4X) to each transferred supernatant. Pipette mix 15x (pipette set to 150 µl).
- If you are mixing the Barcode sample with the cDNA to make the NGS Library, we recommend mixing cDNA samples with the Barcode samples at a ~9:1 ratio (~9 parts cDNA to 1 part barcode/sgRNA reaction).
Processing Cellecta CloneTracker Barcodes (or Guides) Single-Cell Data
For processing Cellecta CloneTracker Barcode single-cell data from the 10x system and assigning CloneTracker barcodes, please use the step-by-step workflow guide on GitHub: https://github.com/Cellecta/CloneTracker_single_cell.
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