title: GSM9247483: mRNA_0min_rep1; Homo sapiens; RNA-Seq
library_construction_protocol: From polysome profiling samples, an aliquot of ribosome fractions representing monosome and polysome were collected followed by digestion with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 ºC for 1 h. 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) at 4 ºC for overnight. 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. 10~200 ng RNAs were mixed with 10 U T4 PNK (NEB), 1 µL homemade Ezra enzyme, 5 U Poly(A) Polymerase (NEB), and 20 U SUPERase·In in Ezra buffer and incubated at 37 ºC for 30 min followed by 70 ºC for 10 min. Ligation was performed for 60 min at 25 ºC by adding a 10 µL reaction mixture (0.5 μM biotinylated 5' end adaptor, 1 × T4 Rnl2 reaction buffer, 20 U SUPERase·In, 15% PEG8000 and 10 U T4 RNA ligase 2 truncated KQ (NEB)). The ligated RNA sample was mixed with 10 µL of pre-washed streptavidin beads (NEB) and incubated at room temperature for 10 min. 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 μM reverse transcription primer, 5 × first strand buffer, 0.1 M DTT, 10 mM dNTP, SuperScript III) followed by incubation at 50 ºC for 30 min. After washing once with 2 × SSC, the 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 primers and 0.025 U Phusion polymerase. PCR was carried out under the following conditions: 98 ºC, 30 s; (98 ºC, 5 s; 68 ºC, 15 s; 72 ºC, 20 s) for 14 cycles; 72 ºC, 2 min. PCR products were separated on a 8% polyacrylamide TBE gel (Invitrogen). DNA products with the expected size around 180 bp were excised and recovered from DNA elution buffer (300 mM NaCl, 1 mM EDTA). After quantification by Agilent BioAnalyzer DNA 1000 assay, equal amounts of barcoded samples were pooled and sequenced using NextSeq 500 (Illumina).