title: GSM8001683: mTSC,ezrmut,rna,3; Mus musculus; RNA-Seq
library_construction_protocol: For RNA-seq experiments : TSCs wild-type and CRISPR-mutated ezr cells were grown separately in wells of a 6-well plate and harvested at 80% confluency (~2e6 cells) using 500 μL of TRIzol reagent (Life Technologies Corp-15596026). For RNA extraction, the lysate was quickly thawed at 65°C, cooled on ice for 5 minutes, and vortexed. Then, 100 μL of chloroform was added per 0.5 mL of TRIzol lysis reagent, shaken vigorously for 15 seconds, and incubated for 3 minutes. The sample was centrifuged at 4°C and 7000 x g for 25 minutes, and the upper colorless aqueous phase was transferred to a new tube. To this, 250 μL of isopropanol was added, incubated for 10 minutes at 4°C, and centrifuged at 4°C and 12,000 x g for 10 minutes. The total RNA precipitate formed a white gel-like pellet at the bottom of the tube, which was washed with 75% ethanol, air-dried for 5-10 minutes, and resuspended in 20 μL of RNase-free water. DNase treatment was performed using the TURBO DNase kit per the manufacturer's instructions: adding 1 ul of TURBO DNase & 2 ul of DNase buffer to the dissolved RNA and incubating at 37 degrees for 30 min. To inactivate TURBO DNase, RNA samples were extracted with phenol/chloroform (Sigma-P3803). After the total RNA pellet dissolved again in 20 ul of RNase-free water. Then, incubate the sample in a water bath or heat block set at 55-60°C for 10-15 minutes. For RNA-seq,The RNA concentration was determined using a NanoDrop spectrophotometer, and The RNA integrity was checked on a 2100 Bioanalyzer using an Agilent RNA 6000 Nano Kit. mRNA-stranded libraries were prepared using a TruSeq poly-A Stranded mRNA Library Prep Kit and sequenced on an Illumina NextSeq 2000 P2 platform with 2 x 100 bp single-end reads. For RNA-seq: mRNA-stranded libraries were prepared using a TruSeq poly-A Stranded mRNA Library Prep Kit For Nascent RNA-seq, we used TT-Seq method as described: https://www.protocols.io/view/transient-transcriptome-sequencing-experimental-pr-3byl42y22vo5/v1; For trophoblast stem cells: approximately 10 million cells were seeded in a 100 cm dish (~80% confluency), and 500 µM of 4sU (Sigma, T4509) was added and kept at 37 °C, 5% CO2 for 15 mins. The cells were then collected by adding 4.5 mL of TRIzol lysis reagent (Life Technologies Corp-15596026), incubated for 5 min on ice, flash-frozen, and stored at −80°C. And in the biotinylation of 4sU labeled RNA step, acid-phenol-chloroform (ThermoFisher, AM9720) was used instead of chloroform. For Nascent RNA-seq, Nascent RNA concentration was measured using the Qubit RNA HS Kit, and RNA integrity was analyzed on Agilent Bioanalyzer RNA 6000 Pico Chip and sequenced on an Illumina NextSeq 500  with 2 x 75 bp paired-end reads. For Nascent RNA-seq: TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold Set, which degrades rRNA with the enzyme.