GSM5918650: E14 mESC Si8L1; Mus musculus; OTHER
GSM5918650
Bulk genomic DNA was extracted using the DNeasy kit (Qiagen, 69504) according to the manufacturer's recommendations. Bulk total RNA was extracted using TRIzol reagent (Invitrogen, 15596018) according to the manufacturer's recommendations. For single cell experiments, whole cell lysate was used by soring cells via FACs directly into lysis buffer. 4 µL of Vapor-Lock (QIAGEN, 981611) was manually dispensed into each well of a 384-well plate using a 12-channel pipette. All downstream dispensing into 384-well plates were performed using the Nanodrop II liquid handling robot (BioNex Solutions). To each well, 100 nL of uniquely barcoded reverse transcription primers (7.5 ng/µL) containing 6 nucleotide UMI was added. The reverse transcription primers used here were previously described in Grun et al. with the exception that a UMI length of 6 was used45. Next, 100 nL of lysis buffer (0.175% IGEPAL CA-630, 1.75 mM dNTPs (NEB, N0447S), 1:1,250,000 ERCC RNA spike-in mix (Ambion, 4456740), and 0.19 U RNase inhibitor (Clontech, 2313A)) was added to each well. Single cells were sorted into individual wells of a 384-well plate using FACS and stored at -80°C. To begin processing, plates were heated to 65°C for 3 minutes and returned to ice. Next, 150 nL of reverse transcription mix (0.7 U RNAseOUT (Invitrogen, 10777-019), 2.33x first strand buffer, 23.33 mM DTT, and 3.5 U Superscript II (Invitrogen, 18064-071)) was added to each well and the plates were incubated at 42°C for 75 minutes, 4°C for 5 minutes, and 70°C for 10 minutes. Thereafter, 1.5 µL of second strand synthesis mix (1.23x second strand buffer (Invitrogen, 10812-014), 0.25 mM dNTPs (NEB, N0447S), 0.14 U E. coli DNA Ligase (Invitrogen, 18052-019), 0.56 U E. coli DNA Polymerase I (Invitrogen, 18010-025), 0.03 U RNase H (Invitrogen, 18021-071)) was added to each well and the plates were incubated at 16°C for 2 hours. Following this step, 650 nL of protease mix (6 µg protease (Qiagen, 19155), 3.85x NEBuffer 4 (NEB, B7004S)) was added to each well, and the plates were heated to 50°C for 15 hours, 75°C for 20 minutes, and 80°C for 5 minutes. Next, 500 nL of glucosylation mix (1 U T4-BGT (NEB, M0357L), 6x UDP-glucose, 1x NEBuffer 4) was added to each well and the plates were incubated at 37°C for 16 hours. Thereafter, 500 nL of protease mix (2 µg protease, 1x NEBuffer 4) was added to each well, and the plates were incubated at 50°C for 3 hours, 75°C for 20 minutes, and 80°C for 5 minutes. Next, 500 nL of MspJI endonuclease mix (1x NEBuffer 4, 8x enzyme activator solution, 0.1 U MspJI (NEB, R0661L)) was added to each well and the plates were incubated at 37°C for 4.5 hours, and then heated to 65°C for 25 minutes. To each well, 280 nL of uniquely barcoded 250 nM unphosphorylated double-stranded Dyad-seq adapters were added. Next, 720 nL of ligation mix (1.39x T4 ligase reaction buffer, 5.56 mM ATP (NEB, P0756L), 140 U T4 DNA ligase (NEB, M0202M)) was added to each well, and the plates were incubated at 16°C for 16 hours. After ligation, uniquely barcoded reaction wells were pooled using a multichannel pipette, and the oil phase was discarded. The aqueous phase was incubated for 30 minutes with 1x AMPure XP beads (Beckman Coulter, A63881), and then subjected to standard bead cleanup with the DNA eluted in 30 µL of water. After vacuum concentrating the elute to 6.4 µL, in vitro transcription (IVT) was performed as previously described in the scAba-seq and scMspJI-seq protocols1,8. RNA enrichment from the IVT product was performed as described previously with the following modifications. The entire IVT product was used for enrichment, 4 µL of 1µM biotinylated polyA primer (5'- AAAAAAAAAAAAAAAAAAAAAAAA/3BioTEG/ -3'), and 8 µL of Dynabeads MyOne Streptavidin C1 beads (Invitrogen, 65001) were used and resuspended in 24 µL of 2x B&W solution after establishing RNase-free conditions. In addition, the supernatant was saved for additional processing. The supernatant from the RNA enrichment process contains unamplified barcoded scDyad-seq DNA molecules. A 1x AMPure XP bead cleanup was performed by incubating the samples with beads for 30 minutes and eluting in 40 µL of water. Samples were then concentrated to 28 µL and nucleobase conversion was performed as described above for bulk M-M-Dyad-seq. Samples were then subjected to four rounds of linear amplification. The first round was the same as described for bulk Dyad-seq. In subsequent rounds, samples were first heated to 95°C for 45 seconds before being quenched on ice. Once cold, 5 µL of amplification mix was added (1x NEBuffer 2.1 (NEB, B7202S), 2 mM dNTPs (NEB, N0447L), 2 uM Linear amplification 9-mer, and 10 U of high concentration Klenow DNA polymerase (3'-5' Exo-) (fisher scientific, 50-305-912)). Samples were then quickly vortexed, centrifuged and the same thermocycler conditions were used as in the first round of linear amplification. After 4 rounds of linear amplification, sequencing libraries were prepared the same way as described for bulk Dyad-seq. Finally, 150 bp paired-end Illumina sequencing was performed on a HiSeq platform. Sequencing metrics and associated metadata for each cell can be found in Supplementary File 1.