GSM9108138: Gast_37_96h_RNA; Mus musculus; RNA-Seq
Gastruloids were generated as described previously. In short, murine embryonic stem cells (mESCs) are washed twice with PBS, followed by a detachment using Accutase (AT-104) for 4 min at 37C, 5% CO2. Cells were collected in 4.5 ml of embryonic stem cell medium with 10% fetal calf serum. Cells were centrifuged 200xg, 4min, 4°C and then resuspended in 10 ml PBS, followed by a second spin and PBS wash. Finally, cells were resuspended in 1 ml of N2B27 and counted using a hemocytometer. 40 µl of N2B27 with 300 cells per well were seeded in low adhesion 96 well plates (CLS7007-24EA) and incubated for 48h at 37C, 5% CO2. After 48 hours gastruloids were pulsed with 150uL of N2B27 supplemented with 3uM CHIR99021 (SML1046-5MG). Medium was refreshed using N2B27 every 24 hours for the extend of culture (between 120-144h). 120h gastruloids were harvested using P1000 cut-tips and subjected to single cell sequencing. In short gastruloids were collected in 15 ml tubes, washed once with PBS and incubated with accutase for 15 mins at 37C. Cell suspensions were resuspended using low adhesion tips and centrifuged 200xg, 4min, 4C. For sorting, cells were resuspended in FACS buffer (2% FCS and 0.5 ng/ul DAPI in PBS), passed through a 35 µm filter cap into 5 ml FACS tubes and sorted for GFP+ populations using a BD FACSAria III cell sorter using a 70 µM nozzel. Sorted cells were then spun down 15 min at 400xg, 4°C and then resuspended in BD sample buffer. Unsorted gastruloids were harvested and processed the same way, resuspended in sample buffer and then passed through 35 µm cell filters 3 times. Single cell suspensions were prepared for sequencing using the BD Rhapsody Single-Cell Analysis System with the BD Rhapsody Cartridge Kit (633733). Bead based reverse transcription, amplification and sequencing library production was performed following manufacturer's instructions (BD Biosciences, doc ID: 210967 rev. 2.0).                                                                   Samples for Nanopore direct RNA-seq: Samples for Nanopore direct RNA-sequencing were harvested at 72 h of gastruloid development. Gastruloids were harvested using cut P1000 tips, collected in 15 ml tubes and washed 2 times in PBS+CHX. Supernatant was removed, and organoids were flash frozen in liquid nitrogen. Frozen organoids were then resuspended in ribosome lysis buffer (1x mammalian polysome buffer, 1mM DTT, 1% TritonX-100, 0.5%NP40, 25U/µL Turbo DNase, 100µg/mL CHX), incubated for 5 min on ice, followed by 5 min spin at 20 000 g at 4°C. Supernatant was collected and flash frozen in liquid nitrogen, before being stored at -80 °C before downstream processing.                    Samples for ribosome profiling: Ribosome profiling experiments were performed as previously described, with minor modifications for gastruloids. In short, gastruloids were harvested at indicated time points using cut P1000 tips and collected into 15 ml tubes. Gastruloids were washed 2 times in PBS+CHX, supernatant was removed, and organoids were flash frozen in liquid nitrogen. Frozen organoids were then resuspended in ribosome lysis buffer (1x mammalian polysome buffer, 1mM DTT, 1% TritonX-100, 0.5%NP40, 25U/µL Turbo DNase, 100µg/mL CHX), incubated for 5 min on ice, followed by 5 min spin at 20 000 g at 4°C. Supernatant was collected and flash frozen in liquid nitrogen, before being stored at -80 °C before downstream processing. Samples for RNA degradation: Gastruloids at 96 h of development were treated with 10 µM Actinomycin D (ThermoScientific, 11805017) in N2B27 and gastruloids were harvested after 0, 2 and 4 h of treatment. Gastruloids were harvested at indicated time points using cut P1000 tips and flash frozen. Frozen organoids were then resuspended in ribosome lysis buffer without cyclohexamide (1x mammalian polysome buffer, 1mM DTT, 1% TritonX-100, 0.5%NP40, 25U/µL Turbo DNase), incubated for 5 min on ice, followed by 5 min spin at 20 000 g at 4°C. Supernatant was collected and RNA was purified using the DirectZol RNA kit. Purified RNA was submitted for library preparation and sequencing to Novogene. 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 gastruloids at indicated time points 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.               Nanopore direct RNA-seq: RNA was isolated from ribosome profiling lysate buffer lysates using Direct-zol RNA Kit, according to manufacturer's instruction. Nanopore direct RNA-sequencing library preparation was performed using the Direct RNA Sequencing Kit (SQK-RNA004) according to manufacturer's instructions. Samples were loaded onto MinION flow cells (FLO-MIN114).