GSM5389600: PseudoTime RA 0d SC g2 2; Mus musculus; RNA-Seq
mRNA extraction was performed using Invitrogen Dynabeads mRNA DIRECT Purification Kit (61011) according to the protocol included with the kit. The library for differential gene expression analysis was prepared using 100-1000 ng of mRNA. First, RNA was fragmented using an RNA fragmentation kit (Ambion, AM8740) following the manufacturer's protocols. Fragmented RNA was purified with 1.4x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 10 μl TE buffer (10 mM Tris–HCl, pH 8.0, 0.1 mM EDTA). The resulting fragmented RNA was reverse transcribed using an oligo dT primer: First, 10 μl of RNA were mixed with 2 μl of 50 μM oligo dT primer (Bio-Rad, 1725038) and 1 μl of 25 mM dNTP mix (NEB, N0447S), and samples were incubated for 3 minutes at 65 °C and transferred to ice. The following components were added to the reaction, for a total volume of 20 μl: 4 μl of 5x First-Strand SuperScript III buffer (Invitrogen, 18080044), 2 μl of 0.1 M DTT (Invitrogen, 18080044), 1 μl murine RNase inhibitor (NEB, M0314L), and 1 μl of 200 U/μL SuperScript III RT enzyme (Invitrogen, 18080044). The reaction product (in the form of a cDNA:RNA hybrid) was purified with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 16 μl TE buffer as described above. For second-strand synthesis, 16 μl of the digestion reaction product were combined with 2 μl second-strand synthesis (SSS) buffer and 1 μl of SSS enzyme mix of mRNA Second Strand Synthesis Module kit (NEBNext, E6111S) and incubated at 16 °C for 2.5 hours, followed by 20 minutes at 65 °C. The resulting product was purified with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted with 20.4 μl TE buffer. cDNA edges were repaired using End-It DNA End-Repair Kit (Danyel Biotech, ER81050) by combining the 20.4 μl sample with 3 μl 10X End-repair Buffer, 3 μl 2.5 mM dNTPs, 3 μl 10 mM ATP, and 0.6 μl END-IT enzyme mix at room temperature for 45 minutes, followed by purification with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and elution with 32 μl TE buffer. Klenow-mediated addition of an adenine to the 3' end of the DNA fragments was performed using the Klenow fragment (3'→5' exo-) kit (NEB, M0212L) by combining the 32 μl sample with 5 μl 10X Klenow Buffer NEB 2, 10 μl 1 mM dATP, and 0.6 μl 5 U/μl Klenow (3'-5' exo-) at 37 °C for 30 minutes, followed by purification with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and elution with 8 μl TE buffer. Illumina library adaptors were added to the resulting cDNA fragment using DNA ligase (NEB, M2200S) by incubation with 5 μl 2x Ligase Buffer, 0.5 μl adapter Oligo mix (1:10 in H2O), and 0.5 μl 1 U/ml DNA Ligase at room temperature for 15 minutes. The ligated product was purified with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 11.5 μl TE buffer. The resulting libraries were PCR amplified using standard PE1/PE2 full-length primer mix containing Illumina library indices for multiplexing (sequences are given in Supplementary Table X). Each PCR reaction contained 11.5 μl post-RT cDNA library, 12.5 μl 2x KAPA HiFi HotStart PCR mix (Zotal, KK-KK2601), and 1 μl of 25 μM 2p Fixed (+barcode) and 2p Fixed primer mix (Supplementary Table 3). Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 32 μl TE buffer. A 15-μl aliquot of each resulting library was run in a 2% agarose gel, and size selection for the desired 200-800 bp DNA library fragments was performed using PureLink DNA gel extraction kit (Invitrogen, K210012). Library quality was confirmed by Agilent 2200 TapeStation Nucleic Acids System.  The in vitro transcription sequencing (IVT) library was prepared using 75-100 ng of mRNA, extracted as described above. First-strand synthesis was performed using SuperScript III Reverse Transcriptase (Invitrogen, 18080044) and the T7RTPolyT primer (Supplementary Table X) at 50 °C for 2 hours followed by 15 minutes at 70 °C and 1 minute at 4 °C. To remove unused primers and primer dimers, the reaction product was combined with 20 μl digestion mix containing 5 μl ExoI (NEB, M0293S), 5 μl HinFI (NEB, R0155S), 5 μl ExoI buffer (NEB, B0293S), 5 μl CutSmart buffer (NEB, B7204S), and 30 μl nuclease-free water and incubated for 1 hour at 37 °C and 10 minutes at 80 °C. The reaction product (in the form of a cDNA:RNA hybrid) was purified with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 13.5 μl TE buffer. For second-strand synthesis, the 13.5 μl digestion reaction product was combined with 1.5 μl SSS buffer and 1 μl of SSS enzyme mix from the mRNA Second Strand Synthesis Module kit (NEBNext, E6111S) and incubated at 16 °C for 2.5 hours, followed by 20 minutes at 65 °C. For linear amplification by in vitro transcription, 16 μl of SSS reaction products were combined with 24 μl HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S) reagent mix containing 4 μl T7 Buffer, 4 μl ATP, 4 μl CTP, 4 μl GTP, 4 μl UTP, and 4 μl T7 enzyme mix. The reaction was incubated at 37 °C for 13 hours, and the resulting RNA was purified with 1.3x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted with 20 μl TE buffer. Aliquots of 9 μl of each sample were frozen for backup at -80 °C, 2 μl of each sample was directly analyzed, and the remaining 9 μl were used in subsequent library preparation steps. RNA was fragmented using the RNA fragmentation kit (Ambion, AM8740) by combining 9 μl sample with 1 μl of RNA fragmentation reagent and incubating at 70 °C for 2 minutes. After transfer to ice, 40 μl fragmentation stop mix containing 5 μl fragmentation stop solution and 35 μl TE buffer was added. Fragmented RNA was purified with 1.4x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 10 μl TE buffer. The resulting amplified and fragmented RNA was reverse transcribed using a random hexamer primer (IDT) as follows: First, 10 μl of RNA were mixed with 2 μl of 100 μM PvG748-SBS12-RT random hexamer primer (Supplementary Table X) and 1 μl of 10 mM dNTP mix (NEB, N0447S), incubated for 3 minutes at 65 °C and transferred to ice. The following components were then added to the reaction for a total volume of 20 μl: 4 μl of 5x First-Strand SuperScript III buffer (Invitrogen, 18080044), 1 μl 0.1 M DTT (Invitrogen, 18080044), 1 μl murine RNase inhibitor (NEB, M0314L), and 1 μl of 200 U/µL SuperScript III RT enzyme (Invitrogen, 18080044). Following reverse transcription, the reaction volume was raised to 50 μl by adding 30 μl nuclease-free water, and the resulting cDNA was purified with 1.2x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 11.5 μl TE buffer. Library amplification was performed using 12.5 µl 2x KAPA HiFi HotStart ReadyMix PCR kit (Zotal, KK-KK2601) and 1 μl of 25 μM 2p fixed primers (Supplementary Table X). Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 30 μl nuclease-free water. Aliquots of 15 μl of each library were run in 2% agarose gels, and size selection for the desired 200-600 bp DNA library fragments was performed using PureLink DNA gel extraction kit (Invitrogen, K210012). Library quality was confirmed using the Agilent 2200 TapeStation Nucleic Acids System (Agilent).  For scRNA-seq, encapsulation of cells with Reverse Transcription mix (IGEPAL CA-630- Sigma-Aldrich I8896-50ML , SuperScript III Reverse Transcriptase Invitrogen 18080044, Deoxynucleotide (dNTP) Solution Set Ornat N0446S, D,L-DITHIOTHREITOL Bio-Lab 000448235200, TRIS-HCL  1 M STOCK SOLUTIONS  PH 7.5  B 1L Sigma-Aldrich T2319-1L, RNase Inhibitor, Murine NEB M0314L) and gels with unique molecular identifiers was performed using the FLUIGENT Smart Microfluidics Pump Syste