GSM4485114: Mettl3 KO fetal liver J2 INPUT rep 1; Mus musculus; RIP-Seq
bulk RNA-seq: RNA were extracted from E14.5 LSK cells following QIAGEN RNeasy Plus Micro Kit protocol. CUT&RUN: Nuclei from E14.5 LSK cells were isolated with NE buffer, captured with Bio-Mag Plus Concanavalin A coated beads (Polysciences) and incubated with primary antibody for 2 hours at 4°C. After washing away unbound antibody with wash buffer, protein A-MNase was added to a final concentration of 700 ng/ml and incubated for 1 hour at 4 °C. The nuclei were washed again and placed in a metal block at 0°C. To activate protein A-MNase, CaCl2 was added to a final concentration of 2 mM. The reaction was carried out for 45 min and stopped by addition of an equal volume of 2XSTOP buffer. The protein-DNA complex was released by incubation for 10 min at 37 °C and centrifugation. DNA was extracted by QIAGEN PCR MiniElute Kit. J2-RIP RNA-seq: Fresh E14.5 fetal livers were harvested. One part of the fetal liver was extracted by TRIzol Reagent for input RNA, the other part of the fetal liver was lysed in dsRIP lysis buffer. The fetal liver lysate was incubated with 5 ug SCICONS J2 antibody for 2 hours at 4 ℃. Protein G Dynabeads were washed in dsRIP lysis buffer and added to the lysate to allow J2-RNA complex formation for 1 hour at 4 ℃. Beads were washed four times with dsRIP lysis buffer, and J2 antibody pull-down RNA was extracted with TRIzol Reagent. single cell RNA-seq: Microfluidic devices were infused with PBS and pressurized to remove air bubbles inside the microwells using a manually operated syringe with a closed outlet. E14.5 LSK single cell suspensions with 20,000 cells/50 µl in PBS + 1%BSA were pipetted on the inlet and drawn into the device. Once the channel was completely filled with cell solution, the fluid flow was stopped, and cells were allowed to settle by gravity. Excess cells were washed out with PBS, and mRNA capture beads at concentrations of 30,000–120,000 beads/50–150 µl were similarly loaded into the cell-loaded wells. Microwells were sealed with fluorinated oil (Fluorinert FC-40), the tubing at the outlet was disconnected, and the microfluidic devices were exposed to three freeze-thaw cycles with 5 min freezing (–80°C) alternating with 5 min thawing at room temperature. Following lysis, the microfluidic device was incubated for one hour inside a wet chamber for mRNA capture onto beads. After incubation, beads were collected into 6x saline-sodium citrate (SSC) buffer and washed twice with 6x SSC buffer prior to reverse transcription. Bulk RNA-seq: polyA RNA selection CUT&RUN: NEBNext Ultra II DNA Library Prep Kit J2-RIP RNA-seq: rRNA depletion single cell RNA-seq: Library preparation steps follow the same steps as in the DropSeq and SeqWell methods
GEO Accession
GSM4485114