title: GSM9107556: RNA-Seq_fasting_trans1; Mus musculus; RNA-Seq
library_name: GSM9107556
library_construction_protocol: For Ribo-seq, RNA was extracted using Trizol LS reagent (Invitrogen) followed by ethanol precipitation. The ribosome-protected mRNA fragments (RPFs) were separated on a 15% polyacrylamide TBE-urea gel (Invitrogen) and visualized using SYBR Gold (Invitrogen). Selected regions in the gel corresponding to 25-35 nt were excised and dissolved by soaking in 400 μl RNA elution buffer (300 mM NaOAc pH 5.2, 1 mM EDTA, 0.1 U/μl SUPERase·In) for 10 min at 70 °C. The gel debris was removed using a Spin-X column (Corning), followed by ethanol precipitation. To enrich capped small RNAs, 50 μg of total cellular RNA was fractionated using the RNA Clean & Concentrator kit (Zymo research). Briefly, adjusted RNA Binding Buffer was prepared by mixing an equal volume of buffer and 100% ethanol. Total RNA sample were mixed with 2 volumes of the adjusted buffer and transferred to the column. The mix was then centrifuged at 16,000 g for 30 s, and the flow-through was collected. 1 volume of ethanol were mixed with flow-through and transferred to a new column. The mix were centrifuged at 16,000 g for 30 s, and the flow-through was discarded. The column was washed by RNA Prep Buffer and Wash buffer, and the bound RNA was eluted with 50 μl DNase/RNase-Free water.  The eluted RNAs were dephosphorylated with 50 U of CIP (NEB) in a 100 μl reaction containing 1 × rCutSmart Buffer and 0.5 U/μl SUPERase·In at 37 °C for 1 h, followed by phenol-chloroform extraction and ethanol precipitation. Three-fourths of resulting RNAs were decapped with 0.75 μl of 5 U/μl Cap-Clip Acid Pyrophosphatase (Cellscript) in 1 × Cap-Clip Acid Pyrophosphatase Buffer at 37 °C for 1 h, followed by phenol-chloroform extraction and ethanol precipitation. The remaining one-fourth RNAs were phosphorylated with 20 U of T4 PNK in a 20 μl reaction containing 0.5 U/μl SUPERase·In, 2 mM ATP and 1 × T4 PNK Buffer at 37 °C for 1 h, followed by ethanol precipitation. For Ribo-seq, trans-RNA-seq, and RNA-seq, 14 μl RNAs (10~200 ng) were mixed with 1 μl T4 PNK (NEB), 20 U SUPERase·In in 1 × T4 PNK buffer and incubated at 37 °C for 30 min followed by 65 °C for 20 min. After ethanol precipitation, 10 μl dissolved RNA were mixed with 1 μl homemade Ezra enzyme, 1 μl Poly(A) Polymerase (NEB) and 20 U SUPERase·In in 7 μl Ezra buffer. After incubation at 37 °C for 30 min followed by 65 °C for 20 min, 1 μl of 1 μM 5' end adaptor, 1 μl of 1 μM biotinylated reverse transcription primer, 20 U SUPERase·In were added and incubated at 70 °C for 3 min followed by slowly cooling down (3 °C/min) to 25 °C. The hybridized RNA sample was mixed with 10 μl of pre-washed streptavidin beads and incubated at room temperature for 10 min. Beads were washed and re-suspended in 10 μl nuclease-free water. Ligation was performed for 60 min at 25 °C by mixing beads with a 10 μl reaction mixture (1 × T4 Rnl2 reaction buffer, 20 U SUPERase·In, 15% PEG8000 and 200 U T4 RNA ligase 2 truncated KQ (NEB)). After washing once with 2 × SSC, beads were re-suspended in 12 μl nuclease-free water and mixed with 8 μl cDNA synthesis mixture (5 × first strand buffer, 0.1 M DTT, 10 mM dNTP, RNaseOUT and SuperScript III) followed by incubation at 50 °C for 30 min. After washing once with 2 × SSC, beads were resuspended in 10 μl nuclease-free water and incubated at 95 °C for 2 min, then immediately placed on the ice for 1 min. After placing on magnet stand for 1 min, the supernatant cDNA was amplified by PCR using barcoded sequencing primers. PCR was performed by mixing 1 × HF buffer, 0.5 mM dNTP, 0.25 μM PCR 5primers and 0.025 U Phusion polymerase. PCR was carried out under the following conditions: 98 °C, 30 s; (98 °C, 5 s; 68 °C, 15 s; 72 °C, 10 s) for 12 cycles; 72 °C, 3 min. PCR products were separated on a 8% polyacrylamide TBE gel (Invitrogen). DNA products with the expected size 180 bp were excised and recovered from DNA elution buffer (300 mM NaCl, 1 mM EDTA).