title: GSM4654308: Willd Type, time 10, replicate 1 [Ribosome profiling]; Mus musculus; OTHER
library_construction_protocol: The media was withdrawn and the cells washed with ice cold PBS supplemented with cycloheximide, scraped and pelleted. The cells were then lysed with lysis buffer (20mM Tris-HCl pH7,4, 150mM of NaCl, 5mM of MgCl2, 1mM DTT, 100 g/mL, 1% of Triton and 25U/mL of Turbo DNase I [AM 2238, Invitrogen] in RNase free water) with the help of glass beads by vortexing 5 seconds for 3 times. The lysate was then left 10 minutes on ice and consequently centrifuged at 10'000 rpm for 10 minutes. To proceed we took an amount of RNA corresponding to 8-10 OD260nm. In order to digest the unprotected RNA, RNAse I (AM2294, Invitrogen) at a concentration of 100U/L was added and incubated at 25°C for 15 minutes. 10L of SUPERase In (AM 2694, Invitrogen) at 20U/L were added to stop RNase digestion and the sample loaded onto a sucrose cushion at 34% w/v in a polycarbonate ultracentrifuge tube (349622, Beckman Culture). The sample were then centrifuged at 70'000 rpm for 4 hours at +4°C. The supernatant was discarded and the pellet resuspended in the resuspension buffer of the Maxwell 16 LEV simplyRNA Cells Kit (AS1270, Promega). The RNA was then extracted according to the manufacturer instructions and resuspended in 40l of RNAse free water. In order to select the fragments of interest we pre-run a 15% TBE-Urea polyacrylamide gel (EC6885BOX, Invitrogen) at 200V for 15 minutes in TBE buffer. 5g of each sample were then mixed with the TBE-Urea sample buffer. The RNA was then denatured at 80°C for 90 seconds and loaded onto the gel and run at 200V for 65 minutes. The gel was then stained for 5 minutes with SYBR green (S32717, Invitrogen) and fragments between 28nt and 32nt excised. The bands were transferred into a RNase-free tube, resuspended in 360l of RNase free water and the RNA extracted by crushing the gel with a pestle. Then it was incubated at 70°C for 10 minutes in order to melt the gel. Once melted the sample was loaded into a spin column with filter made of 2 pieces of whatmann paper and centrifuged at max speed for 2 minutes. Consequently, the RNA was precipitated by adding 40L of 3M sodium acetate, 1L of glycoblue (00585590, Invitrogen) and 500L of isopropanol. The precipitation was performed either for 1 hour in dry ice or overnight at -80°C. Afterward the samples was centrifuged at 20'000g for 30 minutes at +4°C. The supernatant was discarded and the pellet washed with ethanol at 70% in RNase free water. It was then furtherly centrifuged at 20'000g for 2 minutes and then air dried for 10 minutes. Eventually, it was resuspended in 26L of RNase free water. The ribosomal RNA was then removed by making use of the Illumina Ribo-zero rRNA removal kit (20020596, Illumina) according to manufacturer's instructions. The RNA left was then precipitated by adding 300L of isopropanol 20L of sodium acetate, 90L of H2O and precipitated for 1 hour in dry ice. Then it was centrifuged again at 20'000g for 30 minutes at +4°C, the supernatant discarded and the pellet washed with 500L of ethanol 70%, centrifuged again at 20'000g for 5 minutes at +4°C and the pellet was air-dried and resuspended in 10L of TRIS-HCl pH8 10mM. 33L of water were added and the samples were incubated at 80 °C for 90 seconds. In order to dephosphorylate the RNA, 5L of T4 PNK buffer, 1L of SUPERase IN and 1L of T4 PNK enzyme (M0202L, New England Biolabs) were added and the mix incubated at +37°C for 1 hour. The enzyme was then inactivated by rising the temperature at up to +70°C. The RNA was then precipitated and washed as previously stated and resuspended in 8,5L of RNase-free water. In order to add the linker, 1,5L of pre-adenylated linker were previously denaturated (0.5g/L) at +80 °C for 90 seconds and then cooled down at RT.  Then it was ligated by adding 2L of T4 RNA truncated ligase buffer, 6L of PEG 8000, 1L of SUPERase In (20U/L) and 1L of T4 RNA truncated ligase enzyme (200U/L, M024L, New England Biolabs), incubated for 2 hours and a half at RT. Then the RNA was precipitated and washed as previously described and resuspended in 10L of Tris-HCl pH8 10mM.  The samples were run on a 15% TBE gel as previously described, the RNA was excised, extracted and precipitated as previously described and resuspended in 10L of TRIS HCl pH 8 10 mM. In order to convert it to cDNA 2L of reverse transcription primer at 1.25M were added along with 4L of 5x first strand buffer, 1L of dNTPs mix (10 mM), 1L of 1M DTT, 1L of SUPERase In (20 U/L), 1L of Superscript III enzyme (200 U/L, 18080093, Invitrogen) and retro-transcribed at +50°C for 30 minutes. The cDNA was then precipitated and washed as previously described and resuspended in 10L of water. The cDNA was then electrophoretically run, excised and precipitated as previously described. The cDNA was circularized adding 2L of circ-ligase buffer, 1mM of ATP and 50mM of MnCl2 and 100 units of CircLigase I (CL4111K, Epicentre). The sample was then incubated 1 hour at +60°C in a thermal cycler and then heat-inactivate the enzyme for 10 min at +80°C and the rRNA depleted once again. After precipitation and wash the cDNA was resuspended in 5L of TRIS HCl pH8 10 mM and was PCR amplified in 100L volume containing 20L of Phusion HF buffer, 2l of dNTPs mix (10 mM), 0.5l of FW primer (100 M), 0.5l of RV barcoded primer (100 M) and 71L of RNase free water, 1l of Phusion polymerase (2 U/l, M05305, New England Biolabs). The PCR was performed with the following program: 1 cycle at 98°C for 30 sec, 14 cycles at 98°C for 10 sec, 65°C for 10 sec, +72°C for 5 sec. Eventually, the samples were run on an 8% (wt/vol) polyacrylamide non-denaturing gel for 40 min at 180V. The gel was then stained and a band at ~180nt was excised, extracted and purified as previously described. Then the libraries obtained were quality checked and sequenced