GSM7679962: mor28iCre-tdTom OMPtTA-tetOP2, GFP+, RNA-seq, rep2; Mus musculus; RNA-Seq
All RNA-seq experiments were performed under RNA clean conditions. For RNA-seq, live cells were sorted into RNase free 1x PBS, pelleted at 600-g, for 5-min, at 4 ̊C, then resuspended in 500-uL Trizol, flash frozen in liquid nitrogen and stored O/N at -80 ̊C. RNA extraction was performed the next day. Trizol suspensions were thawed on ice, 1/5V of 1-bromo-3-chloropropane was added, and tubes were shook vigorously to combine phases. Phases were allowed to separate for 2-min at room temperature and then tubes were centrifuged at 10,500 rpm, for 15-min at 4  ̊C in an Eppendorf centrifuge C5424R. We collected the upper aqueous phase which was transferred to a new tube. 1/2V of Isopropanol and 1-uL of linear polyacrylamide (Sigma Aldrich 56575) were added, tube was inverted to mix, and RNA was allowed to precipitate for 10-min at RT. Tubes were centrifuged for 10-min at 10,500 rpm and 4- ̊C. The supernatant was removed and 1V of 75% EtOH was added to the pellet, which was dislodged by flicking the tube. Tubes were centrifuged for another 5-min, at 10,500-rpm, and 4  ̊C. EtOH was removed and tubes were allowed to air dry for 5-min until the pellet turned clear. Next we added 26- uL of RNase free water, 3-uL of Ambion DNase I 10x buffer and 1-uL of DNase I (AM2222) to remove all DNA, and incubated tubes at 37  ̊C for 30-min. RNA was purified doing a 1.5x AMPure bead cleanup, measured on a nanodrop, and used as the input for library preparation with SMARTER Stranded Total RNA-Seq Kit- Pico Input Mammalian v2 (Takara Bio USA). OMP-tTA>tetO-GFP and gg8-tTA>tetO-GFP libraries were prepared with the TruSeq kit. However, mature OSN samples were compared to both OMP-tTA>tetO-GFP (TruSeq prep) and OMP-IRES-GFP (Takara Bio USA), which label the same neurons, and produced the same results. Libraries were sequenced on either a NextSeq550 or a NextSeq2000 and were sequenced to a targeted coverage of ~25M reads. All RNA-seq experiments were performed with 2-3 biological replicates.