GSM9140741: Ccx_1-Ccx_6, Cc_1-Cc_6, Hi_1-Hi_6; Mus musculus; RNA-Seq
The mouse brains were isolated from the sacrificed mice and washed with PBS, followed by embedding in optimal cutting temperature compound (OCT) and stored at −80 °C before cry sectioning. The embedded surface of fresh frozen mouse brain tissue was trimmed and sliced by using microtome, and the obtained tissue sections were deposited on polydimethylsiloxane (PDMS) membrane slides. Then the sections were fixed with 4% paraformaldehyde briefly, rinsed with H2O to remove OCT, and dehydrated using isopropyl alcohol for 2 min. After that the treated tissue sections were stained with hematoxylin and eosin (H&E) according to previously reported literature37. The processed tissue sections could be directly used for microneedle sampling or stored at -80 °C freezer for further processing. Before tissue sampling, the PCR tubes or plates pre-added with lysis buffer according to the above described. The home-built microneedle capturing system originated from our group was used for tissue micro-sampling. The microneedle capturing system was consisted of four parts including a 3D translation stage for position control, a system control unit, a microscopic camera system for monitoring the sampling process, and a tapered glass capillary tube for tissue capturing operation. Among this, the capillary tube was fabricated by heating and stretching a glass capillary (200 µm i.d., 360 µm o.d.) in micropipette puller instrument (P-1000, Sutter, USA) to produce a tapered tip. The obtained tissue samples were blown into microtubes by connecting a capillary tube with a compatible syringe. Finally, the tissue samples were centrifuged at 500 g for 2 min to ensure them at the bottom of the tube and were directly analyzed or frozen at -20 °C until library preparation. microregion tissue samples were removed from the −80 °C storage and thawed at room temperature for 10 min to facilitate sample lysis and RNA release, then transferred to a preheated PCR thermocycler, incubated for 3 min at 72 °C and immediately placed on ice afterwards. Next, 10 µl of RT-PCR mix containing 1.2 µL dNTP mix (10 mM), 0.252 µL RNase Inhibitor (40 U/µl), 0.1 µL DTT (100 mM), 0.328 µL MgCl2 (279 mM), 0.18 µL dCTP (100 mM), 2 µL Betaine (5 M), 0.368 µL CBTi-TSO (50 µM), 0.172 µL of Superscript Ⅳ / Maxima H (200 U/µL, ThermoFisher Scientific) and 5 µL of KAPA HiFi HotStart Ready Mix (2×, Roche) were added to synthesis full-length dsDNA. The following RT-PCR program was set to: 60 min at 50 °C, 98 °C for 3 min, then 20 cycles of (98 °C for 20 s, 67 °C for 20 s, 72 °C for 6 min). The cDNA concentration was quantified using the Qubit 4.0 fluorimeter, and size distribution was assessed using capillary electrophoresis on a Fragment Analyzer system (Agilent Bioanalyzer). 1 μl unpurified amplified cDNA working dilution (5 ng/µl) of each sample was transferred to a new plate before adding tagmentation reaction mix as described above. Of note, each sample used unique combination barcoding. Samples were incubated for 10 min at 55 °C. Then, all tagmented products were mixed into 1.5- or 2 ml PCR tube, purified with 1.5× AMPure XP beads for termination of the tagmented reaction and collection, followed by elution with 20 µl nuclease-free water.