title: GSM8841823: FVF_WTA3; Mus musculus; RNA-Seq
library_construction_protocol: P4 back skin was processed as previously described (15). Fat was scraped off and the back skin was washed once in cold PBS and then placed in dispase (Corning #354235) dermis-side facing down, incubated for 35 min at 37 °C on an orbital shaker. Next, epidermis was separated from dermis with fine forceps and placed in 4 ml 0.25% Trypsin-EDTA (1×, Gibco; 25200056) for 15 min at 37 °C with orbital shaking. Epidermis was torn into smaller pieces and washed with cold PBS, pipetted vigorously up and down to achieve a single-cell suspension. Suspension was filtered through a 70-µm and subsequently a 40-µm strainer (Corning; #431750, #431751). Suspension was then centrifuged for 10 min at 400 g and resuspended in FACS buffer (PBS + 2% chelexed FBS (FBS(-)) + DAPI). P60 back skin was processed as previously described (15). Back skin was harvested with surgical scissors and placed dermis-facing up on a styrofoam rack with pins. Fat was scraped off with a scalpel. Fat-free skin was then washed, dermis-side facing down in 1× PBS. Skin was then placed in the same orientation in 0.5% Trypsin-EDTA (10×, Gibco; 15400054) and incubated at 37 °C on an orbital shaker for 25 min (females) or 50 min (males). Using a glass microscopy slide, skin was secured to the bottom of the dish and scraped again with a scalpel until it started to diverge. Excess cold PBS with 2% FBS(-) was added to neutralize trypsin. Cell suspension was strained on ice first through a 70-µm strainer, washed with an additional 15 ml of cold 1× PBS with 2% FBS (-) and then filtered through a 40-µm strainer. Cell suspension was spun down at 400 g for 10 min and resuspended in FACS buffer. P4: a total of 18 injected P4 mice were collected and assessed in 7 independent single-cell RNA sequencing runs. P60: a total of 7 injected mice were collected and assessed in 8 independent single-cell RNA sequencing runs. Library construction protocol for scRNA-seq: EGFP-positive cells were sorted on a BD FACSAria III using a 70-µm nozzle and processed for single-cell capture with a BD Rhapsody Single-Cell Analysis System using BD Rhapsody Cartridge Kit (#633733). For P4, 7 separate cartridges with an average of 76,613 cells were loaded. For P60, 8 cartridges with an average of 68,171 cells were loaded. Reverse transcription from beads and sequencing library production was carried out according to the manufacturer's instructions (BD Biosciences, doc ID: 210967 rev. 1.0). Library construction protocol for RNA-seq: RNA was isolated from lysates from nontargeting control and Gm10076 C7 keratinocytes as described in the Ribosome profiling section using TRIzol®  LS Reagent (Invitrogen, 10296028) and column purified with a Direct-zol RNA MiniPrep kit (Zymo Research, R2052). Libraries were prepared and sequenced at Novogene. Library construction protocol for Other:  As previously described, the sgRNA-containing region was amplified from the BD Rhapsody beads using a nested KAPA Polymerase reaction. In the first polymerase reaction (PCR1), the Rhapsody beads from each separate cartridges were resuspended and distributed into 4 separate 50 µL reactions  (100 µl KAPA HiFi HotStart ReadyMix (Roche 07958935001), 6 µl forward primer (5′-ACACGACGCTCTTCCGATCT-3′, 10 μM), 6 µl reverse primer (5′-TCTTGTGGAAAGGACGA-3′, 10 μM), 12 μl Bead RT/PCR Enhancer reagent from BD Biosciences and 72 μl nuclease-free water for a total volume of 200 μl). PCR1 conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 25 cycles of denaturation 95 °C for 30 s, annealing 53 °C for 30 s, extension 72 °C for 20 s and a final extension of 10 min at 72 °C. The 4 x 50 µl PCR1 product was pooled, and beads were removed using a magnet. Amplicons were cleaned up using Agencourt AMPure XP beads (Beckman Coulter, A63881) following manufacturer's instructions. 3 μl of cleaned up PCR1 was used as template for PCR 2 (25 µL KAPA HiFi HotStart Ready Mix, 2 μl forward primer (5′-ACACGACGCTCTTCCGATCT-3′, 10 μM), 2 μl reverse primer (CAGACGTGTGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA*C*C*G-3′, 10 μM), 18 μl nuclease-free water) for a total volume of 50 μl. PCR2 conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 10 cycles of denaturation 95 °C for 30 s, annealing 60 °C for 3 min, extension 72 °C for 60 s, and a final extension of 5 min at 72 °C. Agencourt AMPure XP beads were used to clean up PCR2 product according to the manufacturer's instructions  before the indexing PCR. Indexing PCR was performed according to BD Biosciences 'mRNA Targeted Library Preparation' protocol (doc ID: 210968 rev. 3.0). Library construction protocol for Ribo-seq: Ribosome profiling was conducted with some adjustments of the original method (64, 65). For RNA digestion, cell lysates were incubated with 1 U/µg of RNase 1 (Epicentre/Lucigen, 10U/µL) for 45 minutes at room temperature. Reaction was stopped by adding 4 µL of SUPERase Inhibitor (20 U/µL, SUPERase-InTM RNase Inhibitor Thermo Scientific AM2694). To eliminate free nucleotides and isolate ribosome-protected fragments, the RNase I-treated samples were processed using Amersham MicroSpin S-400 HR columns (Cytiva, 27514001). RNA extraction was performed utilizing TRIzol® LS Reagent (Invitrogen 10296028) in combination with the Direct-zolTM RNA MiniPrep kit (Zymo Research, R2052). Total RNA concentration was measured using QubitTM RNA broad range kit (Invitrogen, Q10210). Ribosome footprints were excised and extracted from a 15% TBE-UREA gel (Invitrogen, EC68852BOX) using the 20nt, 22nt, 27nt, 30nt and 60nt oligonucleotides mix as markers (1 µL of 10 µM dilution in 19µL RNase-free H2O). Extraction was performed overnight with RNA extraction buffer (300mM NaOAc pH=5.5, 1mM EDTA, 0.25% v/v SDS). T4 PNK (NEB, M0201S) end-healing reaction was performed on the ribosome footprints for 1 hour at 37˚C. Linker ligation reaction followed using T4 RnI truncated K227Q (NEB, M0351L) in combination with 20 µM of preadenylated linker for 3 hours at 22˚C (50% w/v PEG-8000, 10X T4 RNA ligase, preadenylated linker, T4 RnI truncated K227Q). Ribosomal RNA was depleted using biotinylated rRNA depletion oligos (2 µM) by mixing 10 µL of sample with 10 µL of oligo mix and incubated for 2 minutes at 80˚C, followed by a gradual decrease in temperature to 25˚C. MyOne Streptavidin dynabeads (Life Technologies, 65001) were used to recover the samples. Reverse transcription was performed on the Ribosomal RNA-free samples using SuperScriptTM IV Reverse Transcriptase (Invitrogen, 18090050). Footprints were excised and extracted from a 15% TBE-UREA gel. Extraction was performed overnight with DNA extraction buffer (10mM Tris-HCl pH=8, 300mM NaCl, 1mM EDTA). cDNA was circularized using CircLigase II (100 U/µL, Biosearch, CL4115K) for 1 hour at 60˚C. cDNA was amplified using Phusion High-Fidelity DNA polymerase (Thermo Scientific, F530S) using an individual index per sample. 6-12 cycle amplicons were visualized using an 8% TBE non-denaturing polyacrylamide gel to determine optimal cycle number. PCR was carried out with 7-9 cycles and analyzed on an 8% TBE non-denaturing polyacrylamide gel (Invitrogen, EC61252BOX). DNA was excised from gel and extracted using DNA extraction buffer by incubating overnight with gentle shaking. DNA was recovered and precipitated in 10 mM Tris-HCl pH=8. DNA was quantified using Qubit TM 1X dsDNA High Sensitivity Assay (Invitrogen, Q33230) and quality was verified using the Bioanalyzer High Sensitivity DNA kit (Agilent, 5067-4626). Samples were pooled and prepared for sequencing.