MinION sequencing; Transposable element expression at unique loci in single cells with CELLO-seq
Transposable element expression at unique loci in single cells with CELLO-seq
For harvesting and generating single cell suspensions, human iPSCs were harvested using Accutase (Millipore, product code SCR005). Just prior to FACS sorting, cells were resuspended in 1x PBS and strained (Cell strainer (40uM), Corning #CLS431750-50EA). Single cells were sorted into 96-well plates (Framestar #4TI-0960) containing 2 µl lysis buffer with 1U/µl Rnase Inhibitor (RRI), ERCC spike in mix at 1 in 20 Mio dilution (Ambion Cat #4456740). Plates were sealed (Thermo Fisher Scientific #AB0626) immediately spun down and frozen at −80 °C until further processing.  no growth protocol  Human-induced pluripotent stem cells were thawed and cultured under feeder-free conditions on vibronectin coated 15 cm tissue culture treated plates (Corning) in complete medium (500ml Essential 8 medium ((Life Technologies #A1517001), 10 ml Essential 8 supplement, 5ml Penicillin/Streptomycin (Invitrogen #15140122)). Cells were media changed every day except for the day of passaging. Cells were incubated at 37°C, 5% CO2 and routinely passaged using 0.5 mM EDTA (LifeTechnologies #AM9260G).  no treatment After MinION sequencing, we basecalled the raw ONT reads using guppy3.5.2 and then preprocessed the reads using the sarlacc pipeline (Fig S1D). We first performed adapter quality control, where we used minimap2 to align the 250 bp sequence on either end of the read against dt-oligo and TSO template sequence to identify the adapters at either end of the full-length cDNA, followed by filtering for reads with both adapters present44. We then demultiplexed the samples based on the sample barcodes by grouping barcodes that have a Levenshtein distance below the grouping threshold. We checked for internal adapters, which could arise from blunt ligation during ONT library preparation, and used porechop to split the reads if more than 20% of reads had internal adapters. Otherwise we filtered out reads with internal adapters present45. To reduce the search space for the UMI grouping, we performed pre-grouping by mapping the reads to the relevant transcriptome (defined above), ERCC spike-ins as well as unique genome repeat locations of mouse and human UCSC repeatmasker track with minimap2. We grouped the reads by their UMI sequence within each pregroup and either errorcorrected the reads by taking the consensus sequence from multiple sequence alignment of up to 50 reads in the UMI group, or by picking a random read from the UMI group in deduplication mode.  After MinION sequencing, we basecalled the raw ONT reads using the high-accuracy model implemented in Guppy v3.5.2 and then preprocessed the reads using the sarlacc pipeline (Fig S1D). We first performed adapter quality control, where we used minimap2 to align the 250 bp sequence on either end of the read against dT-oligo and TSO sequences to identify the adapters at either end of the full-length cDNA, followed by filtering for reads with both adapters present55. We then demultiplexed the samples based on the sample barcodes by grouping barcodes that have a Levenshtein distance below the grouping threshold. We checked for internal adapters, which could arise from blunt ligation during ONT library preparation, and used Porechop 56 to split the reads if more than 20% of reads had internal adapters. Otherwise we filtered out reads with internal adapters present. To reduce the search space for the UMI grouping, we performed pre-grouping by mapping the reads to the relevant transcriptome (defined above), ERCC spike-ins as well as unique genome repeat locations of mouse and human UCSC repeatmasker track with minimap2 55. We grouped the reads by their UMI sequence within each pregroup and either errorcorrected the reads by taking the consensus sequence from multiple sequence alignment of up to 50 reads in the UMI group, or by picking a random read from the UMI group in deduplication mode.  We performed CELLO-seq for these libraries. Before starting with reverse transcription, we first eliminated any secondary structure in the RNA. We added a 2.2 µl mastermix of 1 µl (1mM) dT-oligo (IDT), 1 µl (1µM) dNTPs (25 mM each, Thermo Fisher Scientific #R0182) as well as 0.2 µl (20U/µL) of RNAse inhibitor (Superase In, Thermo Fisher Scientific, #AM2694) and incubated the mix at 72°C for 3min before returning to ice. The reverse transcription was performed in 10 µl preparing a 5.8 µl mastermix with 1 µl  of 200U/µl Superscript IV Reverse Transcriptase (Invitrogen #18090050), 2 µl of 5x Superscript IV RT buffer, 1 µl of 5M betaine, 0.1 µl of (100µM) template switch oligo (IDT), 0.5 µl of 100mM DTT, 0.1 µl ET SSB (NEB #M2401S) and 1.6 µl nuclease free H2O (DNase/RNase-Free Distilled H2O, Thermo Fisher Scientific #10977-049). cDNA synthesis and template switching were performed for 10 min at 57 °C and 120min at 42 °C. After RT we digested all residual oligos using adding 5 µl mastermix of 4 µl nuclease free H2O (DNase/RNase-Free Distilled H2O, Thermo Fisher Scientific #10977-049), 0.5 µl of Exonuclease I (NEB #M0568) in 0.5 µl of 10x Cutsmart buffer (NEB # B7204S). We incubated the reaction at 37 °C for 1h, followed by 95 °C for 3 min to fully denature both the exonuclease and cDNAs. Before performing the splint ligation, we annealed the splint oligos in 1 µl of 10x Oligo hybridisation buffer (500 mM NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0), 1 µl of 100 µM top splint (IDT) and 1 µl of 100 µM bottom splint (IDT) diluted in 7 µl nuclease free H2O (DNase/RNase-Free Distilled H2O, Thermo Fisher Scientific #10977-049). The reaction was annealed at 95 °C for 1m following 20 s at 95 °C and decreasing temperature by 1 °C every cycle, this step was repeated 80 times and subsequently kept at 4 °C until further processing.   The splint ligation reaction was performed by preparing a 5 µl mastermix that consisted of 0.5 µL Hifi Taq Ligase (NEB #M0647S), 0.5 µl 10x Taq ligase buffer, 1µl of 10 µM annealed splint oligo, 0.25 µL NAD+ (NEB #B9007S) and 2.75 µl nuclease free H2O (DNase/RNase-Free Distilled H2O, Thermo Fisher Scientific #10977-049). The ligation was performed at 55 °C for 1h. Before clean-up we performed a Proteinase K digestion, as the beads were sticky with leftover protein. We digested the proteins with Proteinase K (NEB #P8107S) for 5 min at 50 °C. Barcoded cDNA was then pooled in 2 mL DNA LoBind tubes (Eppendorf #0030108051) and cleaned up using AMPure XP beads at a ratio of 0.5X volume. Purified cDNA was eluted in 12.75 µl H2O. 0.75 µl of the purified DNA was used to perform a proxy qPCR to avoid over-amplification. For the qPCR we used a 25 µL reaction with 12.5µl of 2x KAPA HiFi Uracil+ hot start polymerase mix (Roche # KK2800), 1.25µl of 10 µM forward and reverse PCR primers (IDT), 5µl of 5M betaine, 0.5 µL ET SSB, and 1.25µl of 2x EvaGreen (Biotium #31000-T). The qPCR was performed for 3 min at 90 °C for initial denaturation followed by 29 cycles of 20 s at 90 °C, 30 s at 65 °C, 10 min at 72 °C for 40 cycles. We then used four times fewer cycles than indicated by the qPCR to lead to logarithmic phase of amplification and prepared a PCR reaction of 50 µl PCR master mix consisting of 25µl of 2x KAPA HiFi Uracil+ hot start polymerase (Roche # KK2800) mix, 2.5µl of 10 µM forward and reverse PCR primer, 10µl of 5M betaine, 0.5 µL ET SSB. The PCR was cycled as given: 3 min at 90 °C for initial denaturation followed by 29 cycles of 30 s at 90 °C, 15 s at 65 °C, 10 min at 72 °C, for 25-29 cycles, depending on the qPCR results. These high cycle counts are required to yield the high cDNA inputs (>500 ng) required for optimal ONT sequencing. Final elongation was performed for 10 min at 72 °C. Following amplification, all samples were purified using AmpureXP beads at a volumetric ratio of 0.5X with a final elution in 13 µl of H2O (DNase/RNase-Free Distilled H2O, Thermo Fisher Scientific #10977-049). The cDNA was then quantified using the Qubit High Sensitivity dsDNA Assay Kit (Thermo Fisher Scientific, #Q32851). Size distributions were checked on High-Sensitivity DNA chips (Agilent 2100 Bioanalyzer, #5067-4626).
Experimental Factor: cell type
induced pluripotent stem cell
Experimental Factor: single cell identifier
human_iPSCs_cell_43_longread