Illumina HiSeq 2500 sequencing; Bulk RNA sequencing from sorted VChIs
Bulk RNA sequencing from sorted VChIs
Single-nuclei tissue dissociation: To obtain samples for RNA-seq, whisker-deprived and control mice were sacrificed at day 30 by isoflurane anesthesia followed by cervical dislocation. The left and right barrel cortices were dissected and subjected to the NuNeX procedure; tissues were dissected in ice-cold HBSS (Sigma-Aldrich H6648) and immediately transferred to 1 ml of 10% formalin solution (Sigma-Aldrich HT501128) and incubated at RT for 25–30 min. Samples were then washed in 1xPBS, chopped with a scalpel, and homogenized with a Dounce tissue grinder (Sigma-Aldrich, D9063). The homogenate was passed through a 40 μm cell strainer (BD Falcon) and pelleted by centrifugation at 900 g for 5 min. Cells were resuspended and stained with DAPI (Santa Cruz) for 10 min. All dissociation and staining steps were performed on ice.VChI FACS enrichmentTo enrich barrel cortex VChIs, we sorted cells based on DAPI and tdTomato signals. We combined cells from several mice to reach roughly 200 VChIs per pool, ensuring that each pool was derived from the same mouse strain, treatment condition, sex, and the same hemisphere. To ensure reproducible experimental conditions, we limited whisker plucking to the right side only. RNA was extracted from the pools of sorted cells with a dedicated kit for formalin-fixed tissues (Qiagen, 217504) according to the manufacturer's instructions. See Dataset EV6 for details on sample characteristics and RIN values. Before cell sorting, NeuN antibody was added for staining (1:500, Alexa Fluor®488 conjugated Sigma MAB377X) and incubated for 30 min. Cells were then pelleted by centrifugation at 900 g for 5 min, resuspended in staining buffer, and stained with DAPI (Santa Cruz, DAPI ¦ CAS 28718-90-3) for 10 min. All steps were performed on ice. Cholinergic cells and NeuN-positive/negative cells were sorted through a 70 μm nozzle at a sheath pressure of 70 psi and a drop drive frequency of 90–95 kHz. A highly pure sorting modality (2-way purity sorting) was chosen for both rounds of sorting. The flow rate during the first-round sorting was approximately 5,000 events/s. Samples were collected with BD FACSAria III (BD Biosciences) and analyzed with FCS Express 7 Software (De Novo Software). Perfusion and brain dissection for iDISCO clearing and immunostaining At p30, ChAT-CreXAi14 mice, both control and whisker-deprived, were anesthetized with isoflurane as described above and administered an intraperitoneal injection of 200 mg/kg sodium pentobarbital until nonreactive. Following trans-cardiac perfusion with 1xPBS (5 ml/min) and then 10% formaldehyde in 1xPBS (5 ml/min), mouse brains were collected and further incubated overnight at 4°C in 10% formaldehyde and washed three times with 1xPBS for 30 min. The cortices were dissected and used for iDISCO+ clearing as described initially and further updated in https://idisco.info/idisco-protocol/. Briefly, during the iDISCO+ protocol, we stained the tdTomato expressing cells with an anti-RFP antibody (1:1,200 dilution, Rockland, 600-401-379) for 5 days followed by Alexa-647 conjugated donkey anti-rabbit secondary antibody (1:1,000 dilution, Jackson ImmunoResearch, 711-605-152) for 5 days. Whisker deprivation For whisker deprivation experiments, ChAT-CreXAi14 p7 pups were lightly anesthetized with isoflurane (1–2% by volume in O2, LEI Medical) for 1 min, then quickly removed from the anesthesia chamber and the entire whisker pad plucked individually with tweezers, or slightly pinched without plucking in control mice. After several minutes, once recuperated, treated pups were returned to the home cage and monitored until carried back to the parent cage. For labeling, on day p14 mice were ear-punched on the deprived side.  Sorted cholinergic cells were collected into tubes containing 100 μl PKD (< 1,000 cells). We then folowed the RNeasy FFPE Kit(quiagen) protocol by the manufacturer. RNA content was evaluated using a bioanalyser with the Agilent RNA 6000 Pico Kit.  Library preparation and sequencing800 pg total RNA from each sample pool was subjected to library construction according to the manufacturer's instructions (SMARTer® Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian, Takara Bio). Libraries were barcoded, pooled, and sequenced with a illumina HiSeq2500.