12/2/2023 0 Comments 10x chromium read lengthThis results in final 10x libraries that either represent the 3' end of the transcript (as the 10x Barcode is adjacent to the polyA tail on the 3' end of the transcript) or the 5' end of the transcript (as the the 10x Barcode is adjacent to the TSO and the 5' end of the transcript).Ī schematic diagram comparing the final library construct for the two assay schemes is illustrated below. Downstream of fragmentation, only transcripts containing both (1) a 10x Barcode AND (2) an Illumina Read 2 adaptor, which is ligated on to the cDNA after fragmentation, will be amplified during the Sample Index PCR. A template switching oligo (TSO) is used in both workflows to reverse transcribe the full-length transcript.Īfter amplifying the cDNA, molecules are randomly fragmented under conditions that favor 300-400 bp length fragments. ![]() Both solutions use polydT primer for reverse transcription, although in the 3' assay the polydT sequence is located on the gel bead oligo, while in the 5' assay the polydT is supplied as an RT primer. Sequencing libraries were loaded at 2.1 pM on an Illumina NextSeq500 with 2 × 75 paired-end kits using the following read length: 98 bp Read1, 14 bp I7 Index, 8 bp I5 Index and 10 bp Read2. For a complete list of cellranger vdj command-line arguments, run cellranger vdj -help. Question: What is the difference between Single Cell 3' and Single Cell 5’ Gene Expression libraries?Īnswer: The two assays are similar but capture different ends of the polyadenylated transcript in the final library. Chromium Next GEM Single Cell 5' Library Gel Bead Kit v1.1 reagent (10X. To generate FASTQ files, refer to the instructions on running cellranger mkfastq.For help getting started, try the cellranger vdj tutorial.
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