GSM9110555: VASAseq_Lane_3; Homo sapiens; RNA-Seq
MTs were incubated in 290 U/mg of pre-warmed collagenase type II (Worthington) in HBSS solution (Sigma Aldrich). Single cells were used for single-cell RNAseq. Total RNA for bulk samples was purified using the NucleoSpin RNA Kit  (Bioke') according to the manufacturer's protocol. 10X library: Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3' Reagent Kit, Version 2 Chemistry (10x Genomics) according to the manufacturer's protocol. For bulk RNA-seq: RNA libraries were prepared for sequencing using standard Illumina protocols. For VASA-seq samples: Bulk RNAseq of ECs sorted on day 6 and 27 of CMEC differentiation, and day 27 MT_cmec were performed using VASA-seq. A 96-well plate (Greiner Bio-One, 652250) was filled with 50 μL mineral oil (Merck, M5904) and 1 μL CEL-seq2 primer (Hashimshony et al., 2016) per well with a concentration of 7.5 ng/µl. RNA samples were diluted to 20ng/µl in nuclease free water. Of the dilution, 1µl from each sample was added to different wells of the barcoded 96-well plate. The plates were sealed (Greiner Bio-One, 676090) and spun down at 500 x g for 2 min at 4˚C. Next, library preparation for bulk samples was done as previously described (Salmen et al., 2022) . In short, RNA fragmentation was performed by adding 2 µl of fragmentation mixture per sample consisting of 0.68x First-strand buffer (Invitrogen, Y02321) and the rest, ddH2O. Fragmentation was carried out using a Thermal Cycler (Bio-Rad, T100) set at 85˚C for 3 min with the lid at 105˚C. Next, 2 µl of end repair and poly(A)-tailing mix consisting of 0.12x First-strand buffer (Invitrogen, Y02321), 20 mM DTT(Invitrogen, y00147), 7.5 µM ATP (NEB, P0756S), 0.15 U of E. coli Poly(A) Polymerase (NEB, M0267L), 2 U of T4 PNK (NEB, M0201L) and 8 U of RNaseOUT (Invitrogen, 10777019) was added to each well. Repair and tailing were done at 37˚C in a Thermal Cycler for 1 hour with the lid at 70˚C. For reverse transcription, 2 µl of a mix consisting of 2 mM (each) dNTP (Solis Biodyne, 02-21-00400) and 32 U of SuperScript III (Invitrogen, 18080085) was added to each sample. Reverse transcription was done using a Thermal Cycler set at 50˚C for 1 hour with the lid at 70˚C. The plates were snap-chilled on ice. Next, 11 µl of second strand mix containing 0.23x Second-Strand Buffer (Invitrogen, 10812014), 0.23 mM (each) dNTP, 3.5 U of E. coli DNA Polymerase I (Invitrogen, 18010025) and 0.2 U of RNaseH (Invitrogen, 18021071). Second strand synthesis was done at 16˚C for 2 hours, followed by 85˚C for 20 min with the lid of the Thermo Cycler set to 105˚C. After each aforementioned step, plates were spun down at 500xg for 2 min at 4˚C. The samples were pooled together and the mineral oil was removed. Bead clean-up was performed by adding 1x volumetric ratio of diluted RNAClean XP beads (Beckman Coulter, A63987). The beads were diluted 1:2 in bead binding buffer (20% PEG8000 (Promega, V3011) and 2.5M NaCl). RNA was eluted in 6.4 µl of nuclease-free water. Next, 9.6 µl of IVT mix containing 12mM MegaScript T7 buffer, T7 enzyme, ATP, CTP, GTP and UTP (Invitrogen, AM1334) was added to the pooled sample. IVT was done at 37˚C for 14 hours with the lid set to 70˚C. Following IVT, 6 µl of ExoSAP-IT (Invitrogen, 75001.1.ML) was added and the sample was incubated at 37˚C for 15 min with the lid set to 70˚C. Concentration of aRNA was measured using a Qubit (Invitrogen, Q32853) and adjusted to 100 ng/µl. The rRNA depletion, adaptor ligation, cDNA synthesis, RNA degradation, PCR amplification and final clean-up was used as previously described (Salmen et al., 2022), with a few modifications. The primers used are shown in detail in Table S15. Paired-end sequencing with a read length of 150 bp was performed on the Illumina NovaSeq sequencing platform.