title: GSM4307847: AAV_library_3; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated by using the PicoPure RNA Isolation Kit (ThermoFisher). For RNA isolation of in vitro transduced cells, 1500 cultured NSCs per set were lysed in 100 µl extraction buffer. For isolation of FACS-sorted in vivo transduced cells, batches of 500 cells or less were generated and were lysed in 100 µl extraction buffer. Up to 6 batches (2500 cells) were obtained per set, depending on the cell type (Table S2, Table S3). The cell-containing extraction buffer was incubated for 30 min at 42°C and the lysate was frozen at -80°C to increase the amount of isolated RNA. The cell lysate was mixed 1:1 with 70% ethanol and RNA was extracted according to the guidelines of the PicoPure RNA Isolation Kit (ThermoFisher). RNA was dissolved in 11 µl nuclease-free H2O. The cDNA synthesis was performed as described in 52 by using Locked Nucleic Acid-TSO (Table S1) and by using either 14 cycles for in vitro cultured NSCs, 15 cycles ( >300 cells per batch) or 16 cycles (<300 cells per batch) for FACS-sorted in vivo transduced cells for the cDNA enrichment step. After purification 52 using AMPure XP beads (Beckman Coulter), cDNA was dissolved in 10µl H2O. Barcodes were PCR-amplified by using 10 ng cDNA as input material. Phusion High-Fidelity DNA Polymerase (ThermoFisher) was used according to its manual in combination with 10 mM dNTPs (ThermoFisher) and the Bar_fwd and Bar_rev primers (Table S1). The PCR was performed on a T100 Thermal Cycler (Bio Rad) with the following conditions: initiation for 30 s at 98°C, followed by 35 cycles of denaturation for 10 s at 98°C, annealing/extension for 20 s at 72°C and a final step for 5 min at 72°C. The PCR amplicon was AMPure XP bead-purified (Beckman coulter) 52 with a bead:sample ratio of 0.8:1 in the first round and 1:1 in the second round. Next, 10 ng or 15 ng (library #1 or #3, respectively) of PCR amplicon was used for NGS library preparation with the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina (NEB) for samples from library #1 and the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) for samples from library #3. Multiplexed libraries were generated by following the manual and by using the NEBNext Multiplex Oligos for Illumina (NEB). All multiplexed samples for library #1 and library #3 are listed in Table S2 and Table S3. For sequencing, up to 50% of PhiX were spiked in.