title: GSM3146547: hnRNPL_CLIP; Mus musculus; OTHER
library_construction_protocol: hnRNPL CLIP-seq was performed following the procedures described before with minor modifications (Bao et al., 2018; Licatalosi et al., 2008; Moore et al., 2014; Van Nostrand et al., 2016). Differentiating C2C12 cells (2×107) were washed with PBS and crosslinked by irradiation one time at 254 nm for 400 mJ/cm2 in Stratalinker UV crosslinker (Stratagene). The cells were then collected and flash-frozen in liquid nitrogen and stored at - 80°C. Cells were lysed in 1ml Lysis Buffer (100mM KCl, 5mM MgCl2, 10mM HEPES-KOH pH 7.0, 0.5% NP-40) supplemented with 1mM DTT, 40 U/ml SUPERase In and Complete EDTA free cOmplete protease inhibitors for 15 min on ice with pipetting up and down for several times at intervals. Then, 2 µl of Turbo DNase (AM2238, Thermo Fisher Scientific) was added and the reaction was incubated at 37° for 5 min with rotating at 1000 rpm. Subsequently, 10 µl of diluted RNase A (5 µg/ml, 12091021, Thermo Fisher Scientific) was applied and the reaction was further incubated at 37° for 5 min with rotating at 1000 rpm. Cell lysate was cleared by centrifugation at 15,000 g for 20 min. Save 6 µl of cleared samples as Input sample to later determine immunoprecipitation efficiency and divide the remaining samples into three aliquots. To prepare IP buffer, 2550 μl of ice-cold lysis buffer was supplemented with 600 units SUPERase In, 30 μl of 100 mM DTT and EDTA to 20 mM and 300 μl of cleared cell lysate were then added to the IP buffer. Immunopreciptations were then performed using 10 µg anti-hnRNPL antibody (sc-28726, Santa Cruz), normal IgG control (sc-2027, Santa Cruz) and anti-HA tag (sc-805, Santa Cruz) respectively for overnight at 4°C with rotation. On the next day, 125 µl washed Dynabeads Protein G was added to each reaction and incubated for another 4 hours with rotation. After incubation, 1/10 volume of beads were saved to determine immunoprecipitation efficiency. Beads were collected and washed twice with high salt wash buffer (300mM KCl, 5mM MgCl2, 10mM HEPES-KOH pH 7.0, 0.5% NP-40) then PNK buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.5% NP-40). Cross-linked RNA complexes were dephosphorylated by incubating with Alkaline Phosphatase (NEB) for 20 min at 37°C, followed by treatment with T4 PNK enzyme in acid PNK buffer (70 mM Tris-HCl pH 6.5, 10 mM MgCl2) for 20 min at 37°C. Beads were washed with high salt wash buffer and PNK buffer. Protein-RNA complexes were then divided into two portions. 1/10 volume was subjected to RNA 3' end biotinylation (20160, Thermo Fisher Scientific) following the provider's procedure. Remaining 9/10 volume was used for on-bead 3' RNA linker ligation and incubated in a 30 µl reaction volume containing 50 pmol of 3' RNA adaptor (5'p-rGrArUrCrGrUrCrGrGrArCrUrGrUrArGrArArCrUrCrUrGrArArC-/3'InvdT/) (IDT), 30 nmol of ATP, 30 unites of T4 RNA ligase I (NEB, Ipswich, MA), 20 unites RNase inhibitor and 10% PEG 8000 at 20 ̊C for overnight, shaking at 1200 rpm. After extensive wash, beads (3' RNA adaptor ligated) were 5' ends phosphorylated with T4 polynucleotide kinase (PNK) followed by wash steps. The beads (biotin labeled or 3' RNA adaptor ligated) were then denatured in loading buffer and resolved on a Novex NuPAGE 10% Bis-Tris gel and then transferred to 0.45 µm nitrocellulose membranes for 2h at 60V. The biotin reaction group was then subjected to biotin detection using chemiluminescent nucleic acid detection Module (89880, Thermo Fihser Scientific) to indicate labeled RNA complexes. For western blot analysis to evaluate the IP efficiency, CLIP membranes were probed with anti-hnRNPL antibody for 16h overnight. For sequencing, labeled hnRNPL-RNA bands were excised from the membrane (50-20 kDa above the MW of hnRNPL) and eluted by incubating with PK buffer (100mM Tris-HCl pH 7.4, 50mM NaCl, 10mM EDTA) and Proteinase K at 4mg/ml (Thermo Fisher Scientific) for 30 min at 37°C, then for another 30 min at 37°C with PK buffer, 3.5M Urea and Proteinase K. RNA was subsequently purified using acidic phenol:chloroform (ThermoFisher) and ethanol precipitation. The CLIP RNA was ligated to 5' RNA adaptor (5'-rCrCrUrUrGrGrCrArCrCrCrGrArGrArArUrUrCrCrA-3') (IDT) in a 10 µl reaction volume containing 50 pmol of 5' RNA adaptor, 10 nmol of ATP, 10 unites of T4 RNA ligase I (NEB, Ipswich, MA), 40 unites RNase inhibitor and 10% PEG 8000 at 20 ̊C for 6 hours. Following the ligation, ligated RNA was purified by the acidic phenol:chloroform (ThermoFisher) and ethanol precipitation. The resultant RNA was reverse transcribed with RP1 primer (5'-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3') using Superscript III RT enzyme (Thermo Fisher Scientific). A small portion of cDNA was serial diluted and subjected to test PCR amplification to determine optical PCR cycle. The full-scale PCR amplification was performed using Q5® High-Fidelity 2× Master Mix (NEB) with 12.5 pmol of RP1 primer and RPI-index primers (5' -CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3', in which 'NNNNNN' is an index sequence). The optimal PCR cycle for full-scale amplification is 17. PCR products were PAGE separated and DNA from 140 bp to 350 bp was eluted from PAGR gel. The library was quantified and sequenced in the Illumina HiSeq 1500.
attributes.tag: GEO Accession
attributes.value: GSM3146547