title: GSM9058575: FAC-sorted cells, DNMT1 knockout, rep 1; Mus musculus; RNA-Seq
library_name: GSM9058575
library_construction_protocol: The Cortex samples were collected in cold HBSS (w/ phenol red, w/o calcium, w/o magnesium)/0.65% D-glucose; 4 µg/µL (600 U) of DNAse I (AppliChem GmbH, Germany). After treatment with 0.04% trypsin for 17 min at 37 °C, HBSS was replaced by DMEM with additional L-glutamine and 4.5 g/L D-glucose, 10% FBS and 1% P/S to stop the trypsinization. Subsequently, the cells were pelletized, resuspended and triturated in cold HBSS (w/o phenol red, w/o calcium, w/o magnesium)/0.65% D-glucose, before being filtered through a nylon gauze (pore size 140 µm, Merck, U.S.A.) for FACS. Respective parameters for the procedure using a BD FACS Aria Fusion (BD Biosciences, U.S.A) were defined as follows: 5-laser (FCS, SSC, PE, BV421), 18-color (3-6-2-4-3). TdTomato-positive cells were collected in 100 µL of cold TRIzol™ (Thermo Fisher Scientific, U.S.A.) for subsequent RNA sequencing. Samples were mixed by inversion and stored at -80°C until processing. RNA was isolated from 50,000 cells pooled per genotype. The samples were filled up to 1 ml with TRIzol™ and dounced 25 times in a glass homogenizer using the small-clearance pestle. Samples were incubated for 5 min at room temperature, supplemented with 200 µL chloroform and mixed by shaking vigorously until a homogenous milky solution appeared. Following another incubation at room temperature (until a visible phase separation appeared) samples were centrifuged at 13,000x g and 4°C for 15 min in a tabletop centrifuge with cooling function. For precipitation, the clear upper phase was collected and mixed with 70% ethanol. Samples were bound onto RNeasy mini columns (Qiagen, Germany) and washed according to the instructions in the manual. RNA was eluted with 30 µL nuclease-free water. Prior to library preparation, samples were digested with HL dsDNAseI (ArcticZymes Technologies ASA, Norway) for 10 min at 37°C to remove traces of gDNA. To avoid a digestion of the newly generated cDNA during library preparation, the dsDNAseI was heat inactivated for 5 min at 58°C. Thereafter, samples were subjected to library preparation using the  SMART-Seq Total RNA Pico Input with UMIs (ZapR Mammalian) for Illumina (Takara Bio, Japan). Fragmentation was done at 94°C for 4 min. Illumina adaptors and indices were ligated to cDNA in a PCR reaction with 5 cycles (98°C for 15 s, 55°C for 15 s and 68°C for 30 s). RRNA depletion was performed on the amplified libraries. Final libraries were subjected to an upscale PCR with 15 cycles (98°C for 15 s, 55°C for 15 s and 68°C for 30 s). Amplified libraries were cleaned with magnetic AMPure XP beads (Beckman Coulter, U.S.A.). Library concentration was determined with a Qubit™ 4 fluorometer using the 1x dsDNA HS reagent (Thermo Fisher Scientific, U.S.A.). Library size was assessed on a Tape Station 4200 using the DNA D1000 kit (Agilent Technologies, U.S.A.). Libraries were diluted, equimolarly pooled, denatured, and loaded for clustering onto an Illumina NovaSeq 6000 SP (200 cycles) flow cell (Illumina, U.S.A.) in conjunction with 1% PhiX control library, and run on an Illumina NovaSeq machine (Illumina, U.S.A.) in 75 bp paired-end mode. The 1% PhiX control library was spiked in to improve base calling accuracy.