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| SG-102-Delta-plate2 |
RNA was extracted from PAXgene blood RNA tubes (Swiss Pediatric Sepsis Study, SPSS) utilizing the PAXgene Blood RNA Kit from PreAnalytiX, strictly adhering to the manufacturer's guidelines. The eluted RNA was subsequently concentrated to a 14L volume using the RNeasy MinElute Cleanup Kit from Qiagen. This concentrated RNA was prepared for downstream TCR amplification. Previous research based on high-throughput sequencing (HTS) has shown that functional TRG rearrangements can be found in all blood T lymphocytes including T cells (Sherwood 2011). We therefore focused our analysis on the TRD chains in the whole blood samples. cDNA synthesis was conducted through a template switch anchored reverse transcription PCR (RT-PCR). This employed a primer specific to the human constant delta gene segment (TRDC) with the sequence 5-GTAGAATTCCTTCACCAGACAAG-3, a template-switch adaptor with the sequence 5-AAGCAGTGGTATCAACGCAGAGTACATrGrGrG-3, and the SuperScript II RT enzyme from Invitrogen. The cDNA was then purified using AMPure XP beads by Agencourt. Amplification of the TRD region was achieved through an additional PCR step. We used a TRDC-specific primer with the sequence 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACGGATGGTTTGGTATGAGGCTGACTTCT-3 (adapter sequence italicized) and a primer complementary to the template-switch adaptor with the sequence 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAGCAGTGGTATCAACGCAG-3 (adapter sequence italicized) in combination with the KAPA Real-Time Library Amplification Kit from Kapa Biosystems. Adapters were incorporated to facilitate subsequent sequencing reactions. Another round of purification with AMPure XP beads was followed by index PCR, incorporating Illumina sequencing adapters using the Nextera XT Index Kit. The final PCR product was purified again with AMPure XP beads. HTS of the resulting amplicons containing the TRD sequences was carried out at the GIGA center, University of Liege (Belgium), on an Illumina MiSeq platform with Illumina MiSeq v3 600 cycles kit |
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