GSM9578503: Neuro-2a, NIH/3T3, and mESCs mixture; Mus musculus; RNA-Seq
Cells were collected at roughly  1×10^6 cells/mL, transferred into 15 mL conical tubes, pelleted by centrifugation (100–300 × g for 5–7 min), and washed twice with ice-cold 1X Dulbecco's Phosphate-Buffered Saline (DPBS; Gibco 14190144). Cell number and viability were assessed with a hemocytometer using 0.2% trypan blue staining. The cells were then subjected to an additional centrifugation step, the supernatant was discarded, and the cell pellet was resuspended to achieve a final volume corresponding to 3 million cells per milliliter. Illumina sequencing libraries were prepared for each amplified cDNA aliquot with NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina (NEB, cat. no. E7805S) according to the instructions in the manual, with minor changes. In particular, 5-10 ng of amplified cDNA was used for cDNA fragmentation and transferred to a PCR tube. Nuclease-free water was added to reach the final volume on 13 µL. Fragmentation reaction was assembled on ice by adding 3.5 µL of NEBNext® Ultra™ II FS Reaction Buffer followed by 1 µL of NEBNext® Ultra™ II FS Enzyme Mix. Reaction mix was vortexed and spun-down for 3-5 seconds. The fragmentation was performed for 8 minutes at 37 °C, followed by end-repair and dA tailing for 30 min at 65 °C (lid was set at 75 °C). Fragmented cDNA was purified with 0.6X-0.8X double-size selection using SPRIselect beads and eluted in 17.5 µL with nuclease-free water. Then, 1.5 µM ligation adapter was prepared in advance by mixing 1.5 µL of forward ligation primer (100 µM, /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCAC/3ddC; HPLC purification, IDT) and 1.5 µL of reverse ligation primer (/5AmMC6/GCTCTTCCGATCT; HPLC purification, IDT) with 97 µL of water. To anneal oligos, the solution was heated to 95 °C for 2 min (lid at 100 °C) and cooled at room temperature for 5 min. Next, purified fragmented cDNA was mixed with 15 µL of NEBNext® Ultra™ II Ligation Master Mix, 1.25 µL of ligation adapter and 0.5 µL of NEBNext® Ligation Enhancer. Reaction solution was well mixed by pipetting and incubated for 15 min at 20 °C (lid set at 37 °C). The ligation product was diluted to 100 µL with nuclease-free water, followed by a 0.8X SPRI size selection procedure and eluted in 40 µL with nuclease-free water. Finally, the ligation product was amplified by 11-cyles of PCR: 95 ºC for 3 min, 12-cycles [98 ºC for 20 s, 54 ºC for 30 s, 72 ºC for 20 s], and 72 ºC for 60 s. PCR mix was prepared by mixing 20 µL of ligation product, 2.5 µL of forward indexing primer (Table  S25), 2.5 µL of reverse indexing primer (Table S25) and 25 µL of 2X KAPA HiFi HotStart ReadyMix. Amplified sequencing libraries were purified with 0.6X-0.8X double-size selection using SPRIselect beads and eluted in 20 µL with nuclease-free water.