Single-cell RNAseq library preparation of fragment-seq experiments using Chromium 10X and MULTI-seq

Fragment-seq library construction for murine spleen samples was performed with Chromium 10X. Libraries were generated following the manufacturer’s instructions from Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 protocol (kit component number PN-1000165). In short: cells were resuspended in 0.04% BSA and mixed with a Master mix containing reagents for reverse transcription. The cell suspension was then loaded in GemCode Single-cell Instrument (10X Genomics) together with GemCode Single-Cell 5’ Gel Beads. Cells and beads were fused to generate single-cell Gel Bead-in-Emulsions (GEMs). Within GEMs, cells were lysed and RNA was reverse transcribed. After GEMs were broken and cDNA was cleaned up, using DynaBeads MyOne Silane Beads (Thermo Fisher #37002D) and SPRIselect beads (Beckman Coulter #{"type":"entrez-nucleotide","attrs":{"text":"B23318","term_id":"2508949","term_text":"B23318"}}B23318), cDNA was amplified and cleaned up using SPRIselect beads. Then amplified cDNA was enzymatically fragmented and indexed sequencing libraries were generated by the following steps: end repair, A-tailing, adapter ligation, post-ligation SPRIselect cleanup, and sample index PCR. For MULTI-seq library preparation instructions were followed from McGinnis and Patterson et al. ²⁵. In short: The MULTI-seq primer (according to MULTI-seq protocol instructions) was added in a concentration of 10 µm to the cDNA amplification mix. After cDNA amplification during SPRIselect clean up the non-bound fraction (containing small cDNA fragments) was saved and cleaned up with SPRIselect beads in a higher ratio to enrich for small MULTIseq barcodes. These products were then used for index PCR using the SI-PCR Primer from the 10X kit for the i5 and one of the small RNA TrueSeq index primers for the i7 (Illumina #15004197).