GSM4211909: Fig1-U12IAA-6; Homo sapiens; OTHER
For crosslinking IPs, cells were crosslinked with 0.2% formaldehyde and cells were lysed in RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mM NaCl, 2 µg/ml aprotinin and leupeptin, 1 mM PMSF) and sonicated 3x for 20 seconds. Lysates were precleared for 1 hr at 4˚C with protein A sepharose and then IPs were carried out with antibodies listed in metasheet at room temperature for 90 minutes before 6x washes in denaturating RIPA buffer and then 3 washes with NET2 before on-bead ligation of AG10/11. Ligated RNA was reverse crosslinked with 70˚C incubation for 45 minutes and then extracted with TRIzol per manufacturer's recommendation. For nascent RNA and total RNA librariers, 2.5 µg of RNA was ligated and then extracted with phenol/chlorofom/isoamyl alcohol. For nascent RNA, Click-iT nascent RNA capture kit (Invitrogen) was used on 1.25 µg of RNA according to manufacturer's protocol with the exception of using Superscript III reverse transcriptase (Invitrogen) for cDNA synthesis. RNA adapters containing bar-codes and 10-11 nucleotide randomers were ligated to the 3' ends of total and eluted RNA with T4 RNA ligase (NEB) at 16°C for 16 hours. cDNA was generated from AR-17 primer with Superscript III (Life Technologies). 3' ends were amplified by Q5 High-Fidelity DNA Polymerase (NEB) first, using snRNA gene-specific primers and AR-17 and then with primers D50x and D70x (Illumina). Libraries were purified on AMPure XP magnetic beads (Beckman), and validated by 2200 Tape Station Instrument (Agilent). Libraries were pooled at 4 nM, denatured with NaOH and diluted in HT1 buffer (Illumina) to 7 pM before sequencing with MiSeq Reagent Kit V2 Nano configuration (Illumina).
GEO Accession
GSM4211909