iPS2-multi-seq on early pasage (P3) iPS2-seq targeted hiPSC
We aimed to identify the nature of iPSC and iPSC-neuro clones identified in E-MTAB-14065. We evaluated the combined effect on the epigenome and transcription at the single-cell level. This experiment demonstrated the importance of clonal tracking when performing scRNAseq screenings and allowed us to validate iPS2-multi-seq. Sample preparation for iPS2-multi-seq was performed following the 10X Genomics demonstrated protocol for nuclei isolation (CG000365, Rev D) and the 10X Multiome User Guide (CG000338, Rev F). Cells were detached using Versene and incubated with DNase I (200 U/mL) to reduce clumping, then washed and filtered through a 40 µm strainer (Flowmi). Cell viability and count were assessed using trypan blue staining (Sigma) on a hemocytometer. HiPSC cells P3 transfection pools were kept separately, and nuclei were pooled only prior to loading on the 10X Chromium. After cell lysis using freshly prepared buffer containing 0.1% Tween-20, IGEPAL CA-630, 0.01% digitonin, 1% BSA, and 1U/ml RNAse inhibitor, nuclei were isolated, washed twice with chilled wash buffer, and counted using both trypan blue and 1:1000 YOYO-1 staining (Invitrogen). Final nuclei counts were pooled 1:1 and diluted to 7,000 nuclei/µL before loading into the microfluidic chip. Nuclei were loaded after diluting in 10X Nuclei buffer according to the user guide. Chromatin tagmentation and reverse transcription were performed according to the 10X protocol. Libraries were indexed and amplified: 7 cycles for ATAC index PCR and 6 cycles for cDNA amplification. D5000 Tapestation and dsDNA hi-sensitivity Qubit analyses revealed appropriate fragment sizes and concentrations. The iPS2-seq library was obtained from the cDNA of the 10X gene expression library. Adaptations of the current protocol were necessary to obtain clean iPS2-seq libraries from the multiome cDNA library. Reduced cycle amplification to 11 cycles and increased input material to 200ng was key to this optimization, resulting in libraries with cleaner profiles, confirmed by D5000 Tapestation analysis and improved size distributions following 1.8x–0.7x SPRIselect purification. Libraries were further amplified and indexed using TT indices and purified again using a double-sided size selection.Sequencing was performed on an Illumina NextSeq 1000 using two flow cells P2 Xleap 100 cycles with paired-end dual indexing. ATAC libraries were sequenced at 400 million reads with 650 pM loading concentration and 1% PhiX spike-in (R1: 50 bp; i7: 8 bp; i5: 24 bp; R2: 49 bp). The gene expression library was pooled with the iPS2seq library with a 1:20 ratio, accounting for 380M reads for the gene expression and 20M reads for the iPS2seq library. Final pooled libraries were quantified using Qubit and loaded 650pM and 1% PhiX spike-in (R1: 28 bp; i7/i5: 10 bp; R2: 90 bp). iPS2-seq library generation required further optimization, compared to the original protocol described here, cDNA from 10X multiome as template for iPS2-seq PCR, required reduced cycle amplification and increased input material (up to 200 ng, 11 cycles), resulting in libraries with cleaner profiles, confirmed by D5000 Tapestation analysis and improved size distributions following 1.8x–0.7x SPRIselect purification.