BCR Sequencing was carried out as described previously (37 (link)). Briefly, whole transcriptome amplification (WTA) was performed on the sorted cell-lysates according to the Smart-Seq2 protocol (105 (link)). We then amplified heavy and light chain sequences from the WTA products utilizing pools of partially degenerate pools of V region specific primers (Qiagen HotStar Taq Plus). Heavy and light chain amplifications were carried out separately, with each pool containing pooled primers against human IGHV and heavy chain constant region genes, or human IGLV, IGKV, and light chain constant region genes. Cellular barcodes and index adapters (based on Nextera XT Index Adapters, Illumina Inc.) were added using a step-out PCR method. Amplicons were then pooled and sequenced using a 250×250 paired end 8×8 index reads on an Illumina Miseq System. The data were then demultiplexed, heavy and light chain reads were paired, and overlapping sequence reads were obtained (Panda-Seq) (106 (link)) and aligned against the human IMGT database (107 (link)).
Nextera xt index adapters
Manufactured by Illumina
Nextera XT Index Adapters are a set of oligonucleotide sequences that are designed to be ligated to DNA samples during library preparation for next-generation sequencing. The adapters enable the unique identification of individual DNA samples within a pooled sequencing run.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using nextera xt index adapters
1
High-throughput BCR Sequencing of Sorted Cells
2
High-Throughput BCR Sequencing and Analysis
3
High-Throughput BCR Sequencing and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BCR Sequencing was carried out as described previously (15 (link)). Briefly, whole transcriptome amplification (WTA) was performed on the sorted cell-lysates according to the Smart-Seq2 protocol (52 (link)). Heavy and light chain sequences were amplified utilizing partially degenerate pools of V region specific primers (Qiagen HotStar Taq Plus). Heavy and light chain amplifications were carried out separately. Cellular barcodes and index adapters (based on Nextera XT Index Adapters, Illumina Inc.) were added using a step-out PCR method. Amplicons were pooled and sequenced using a 250x250 paired end 8x8 index reads on an Illumina Miseq System. Data were demultiplexed, heavy and light chain reads were paired, and overlapping sequence reads were obtained (Panda-Seq) (53 (link)) and aligned against the human IMGT database.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!