GSM8561376: bulk_BCR_sequences_skin_technical_rep2_biological_rep2; Mus musculus; OTHER
High-throughput sequencing of the adaptome in the study samples was conducted using the iR-RepSeq+ service provided by iRepertoire, Inc. (Huntsville, AL, US). Next-generation sequencing libraries were generated for each of the 12 samples in replicate, encompassing the BCR heavy chain, with coverage and distinction of IgM, IgD, IgE, IgA, IgG1, IgG2 (IgG2A/IgG2B/IgG2C), and IgG3 subtypes. This technology facilitates amplification of the BCR heavy chain while addressing known sources of errors in AIRR-seq analyses. The sequencing protocol involves the initial utilization of primer pairs for each V-D-J combination, enabling extension with tags for subsequent global amplification of the heavy chains. This amplification process is conducted in a single assay, incorporating unique molecular identifiers (UMIs) during the reverse transcription step to distinguish between individual RNA molecules and minimize the impact of PCR duplicates and sequencing errors. Reverse transcription was performed using Qiagen OneStep RT-PCR mix, followed by selection of first- strand cDNA and removal of remnant primers using SPRIselect bead selection (Beckman Coulter). Subsequently, a second round of binding and extension was carried out with a V-gene primer mix, and SPRIselect beads were employed to purify the first and second-strand synthesis products. Library amplification was achieved using primers specific to communal sites engineered onto the 5′ end of the C- and V-primers employed in the initial synthesis steps. The final constructed library includes Illumina dual index sequencing adapters, a 10-nucleotide UMI, and an 8-nucleotide internal barcode associated with the C gene primer. Amplified libraries were multiplexed and pooled for sequencing on the Illumina NextSeq 1000 platform with one P1 600-cycle kit (300 paired-end reads) at iRepertoire Inc. The resulting immune receptor sequence data covers the second framework region through the beginning of the constant region, including the CDR3 hypervariable region.