10X Chromium Single Cell 3'建
The Chromium Single Cell 3’ Solution V2 chemistry is shown here. Oligo sequence information is taken from The 10x Genomics Technical Note. You can find out all the cell barcodes (16 bp) here: 737K-august-2016.txt. This file is copied from Cell Ranger (using Cell Ranger v2.1.0 as an example) /path/to/cellranger-2.1.0/cellranger-cs/2.1.0/tenkit/lib/python/tenkit/barcodes.
The V3 chemistry gave better sensitivity (detects more genes) comparting to the V2 chemistry. In addition, the oligo beads are modifidied to support feature barcoding. In terms of the 3' gene expression library preparation, there are only a few nucleotide differences for some oligos. The final libary structure is exactly the same, except that the UMI is 10-bp long in V2 but 12-bp in V3. Oligo sequences are provided for both V2 and V3 if they are different. Only V2 library preparation is drawn here.
Adapter and primer sequences:
Beads-oligo-dT:
cDNA Reverse primer:
Step-by-step library generation
(1) mRNA capture using Beads-oligo-dT in the droplets, and reverse transcription using MMLV:
(2) The terminal tranferase acitivity of MMLV adds extra Cs:
(3) Adding TSO for second strand synthesis:
(4) Adding cDNA Forward and Reverse primers to amplify full length cDNA:
(5) Use Fragmentase to fragment cDNA and perform A-tailing:
(6) Add double stranded Illumina Truseq adapter (with a T overhang) for ligation:
Product 1 (I assume the 5' end of TSO is blocked, so the adapter can only be ligated to the cDNA end.
This product is not amplifiable due to the use of the specific primers for amplification, see the next step):
(7) Add Library PCR Primers 1 & 2 to amplify library:
(8) Final library structure:
V2 library:
V3 library:
Library sequencing:
(1) Add Truseq Read 1 primer to sequence the first read (bottom strand as template, sequence 16-bp cell barcode and 10-bp UMI, 26 cycles for V2, 28 cycles for V3):
(2) Add Sample Index sequencing primer to sequence the sample index (bottom strand as template):
(3) Cluster regeneration, add Truseq Read 2 primer to sequence the second read (top strand as template, sequence cDNA, 98 cycles):
