VDJ chain pair sequence
Bowties, Barcodes, and DNA Origami: A Novel Approach for Paired-Chain Immune Receptor Repertoire Analysis
https://repository.asu.edu/attachments/194017/content/Schoettle_asu_0010E_17434.pdf
One exciting application of DNA nanotechnology isthe intracellular capture, barcode linkage, and subsequent sequence analysis of multiple messenger RNA (mRNA) targets from individual cells within heterogeneous cell
ii populations.
DNA nanostructures can be transfected into individual cells to capture and
protect mRNA for specific expressed genes, and incorporation of origami-specific bowtie-barcodes into the origami nanostructure facilitates pairing and analysis of mRNA from individual cells by high-throughput next-generation sequencing.
DNA origamiがindividual cellに感染し、mRNAをキャッチする
One major area of interest for application of DNA nanotechnology is for sensing
the intracellular environment [334-336] and/or capturing and sequencing mRNAs from
individual cells [263, 271].
Recent advancements in microporation technology (the use of µL volume
electroporations) have shown high transfection efficiencies (>93%) as well as high cell
viabilities (>86%).
MicroporationでDNA origamiを細胞に入れる。
unctional Interrogation and Mining of Natively Paired Human VH:VL Antibody Repertoires,
#chainpair
#chainpair
RT-mix
Table 5 | RT-PCR solution.
Component
Amount per
reaction (ml) Final amount
Common primer mix (containing both oligos from Table 1, 10 μM (each)) 115 400 nM (each)
Constant region primer mix (containing all oligos from Table 2, 10 μM (each)) 11.5 40 nM (each)
VH FR1 overlap extension primer mix (containing all VHx oligos from Table 3, 12.5 μM (each)) 9.2 40 nM (each)
VL FR1 overlap extension primer mix (containing all VLx and VKx oligos from Table 3, 7.7 μM (each)) 14.9 40 nM (each)
Ultrapure BSA (50 mg/ml) 29 1.45 mg
qScript fast one-step RT-PCR master mix (4×) 719 1×
SUPERase·In RNase inhibitor (20 U/μl) 115 2,300 U
qScript one-step reverse transcriptase (20×) 144 1×
H2O Fill to 2,875 —
https://doi.org/10.1016/j.jbiosc.2019.01.020
Ultrasonically-guided flow focusing generates precise emulsion droplets for
high-throughput single cell analyses
超高速にドロップレットを作る方法ソニケーションを使っている BCR pairもしている
https://sci-hub.tw/10.1126/scitranslmed.aac5624
High-throughput pairing of T cell receptor
a and b sequences
これは各ウェルでバーコードを分けている
どういうことかわからない
TCRまたはBCRの二つの鎖をつなげて、ペアの情報を保ったままシーケンスする
TCRまたはBCRは二量体で構成されており、このペアの情報が必要である。それぞれ別々のmRNAから翻訳されるため、Dropletで細胞を閉じ込めてから二つのmRNAをつなげることができれば、dropletを壊した後でもペアの情報が保てる。今回はそのような論文を紹介する。
TCR
Pairing of T-cell receptor chains via emulsion PCR
この論文は、
BCR
In-Depth Determination and Analysis of the Human Paired Heavy and Light
Chain Antibody Repertoire
https://media.nature.com/original/nature-assets/nm/journal/v21/n1/extref/nm.3743-S1.pdf
https://drive.google.com/drive/u/0/folders/1_pLCM2VeLinZWXeG__YI2k2adw3hYbtJ
In-depth determination and analysis of the human
paired heavy- and light-chain antibody repertoire
T cell はとてもLow efficiency
BCRのPair
Recombinant human B cell repertoires enable
screening for rare, specific, and natively paired
antibodies
Ultra-high-throughput sequencing of the immune
receptor repertoire from millions of lymphocytes
In-depth determination and analysis of the human
paired heavy- and light-chain antibody repertoire
Pairing of T-cell receptor chains via emulsion PCR
TCRのペアーのシーケンス
support information
https://drive.google.com/file/d/1mJoFU5lsHu1Mzk21_tS-hLWtWvH6atzY/view
プライマー
・BC_synth_rev
・AC_synth_rev
・BV7_for
・AV_for_uni
・13 TRAV primers
・・AV14_for
AC26-1/4_for