GSM8742394: fig4b_control_7sk.rep3; Homo sapiens; ncRNA-Seq
RNA was isolated using TRIzol (Thermo Fisher) per manufacturer's recommendation. RNA was subsequently treated with DNase I (1 U/µl; Zymo research) in DNase buffer (10 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2) at 25˚ C for 15 minutes, followed by extraction with phenol:chloroform:isoamyl alcohol and ethanol precipitation. For RNA immunoprecipitations, cells were harvested by scraping into ice cold PBS and flash frozen in liquid nitrogen. Cells were resuspended in ice-cold isotonic lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 80 U/mL RNaseOut (Thermo Fisher), protease inhibitor tablet/10 mL (Roche)) and incubated with rotation for 20 minutes at 4°C. Cellular debris was pelleted at 13,000 g for 15 minutes at 4°C. The supernatant was briefly pre-cleared using Protein G beads (Thermo Fisher, 10004D) for 10 minutes at 4°C with rotation. Beads for immunoprecipitation were washed with bead wash buffer (PBS pH 7.4, 0.1% Tween 20) and subsequently incubated with anti-La antibody (Santa Cruz Biotechnology, sc-80656), anti-SRP54 antibody (Invitrogen, MA5-34835), rabbit IgG (Cell Signaling Technology, 2729), or mouse IgG1 (Cell Signaling Technology, 5415) antibody for 1 hour at 4°C with rotation, then washed with bead wash buffer. Sample protein was quantified with a BCA assay (Thermo Fisher). 1 mg of protein per sample was diluted 5-fold with ice-cold detergent dilution buffer (isotonic lysis buffer without Triton X-100) and incubated with antibody-bead complexes for 1 hour at 4°C with rotation. Samples were washed 5 times with ice-cold immunoprecipitation wash buffer (PBS pH 7.4, 0.1% Tween 20, 150 mM NaCl) at 4°C. RNA from input and immunoprecipitation material was extracted with TRIzol (Thermo Fisher) according to the manufacturer's recommendation. For SLAMSeq, samples were treated with either iodoacetamide (IAA) or dimethylsulfoxide (DMSO) for 15 minutes at 50°C prior to 3' adaptor ligation. Gene-specific 3'-end sequencing libraries were prepared as described above. RNA adapters containing barcodes and 10- to 16-nt randomers (AG10/AG11/AG15/AG16) were ligated to the 3′ ends of 2 μg of extracted RNA in a 10-μL reaction containing 1 μL of 10xT4 RNA ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM DTT at pH at 25°C), 0.2 mg/mL bovine serum albumin (BSA), 2 μM AG10 or AG11, 1 mM ATP, 10 U of T4 RNA ligase (NEB), and 40 units of RNaseOUT (Invitrogen) for 16 h at 16°C and extracted with phenol/chloroform/isoamyl alcohol. cDNA was generated using SuperScript III (SSRT III; Thermo Fisher) in a 20-μL reaction using 0.5 μM linker-specific primer AR-17. For sequencing, snRNA cDNA 3′ ends were amplified in 16–30 cycles of polymerase chain reaction (PCR) with small RNA gene-specific forward primers and AR17 primer using Q5 DNA polymerase (NEB), and then eight cycles with primers D50x and D70x. Libraries were sequenced on an Illumina MiSeq platform.