title: GSM4152109: U1 SHAPE-MaP; Homo sapiens; OTHER
library_construction_protocol: [SLNCR MaP]  From each sample of RNA from WM1976 in-cell and cell-free probing experiments, 1-3 µg were subjected to mutational profiling (MaP) reverse transcription (Siegfried et al., 2014; Smola et al., 2015b) using a primer specific to SLNCR. The cDNA generated was buffer exchanged over Illustra microspin G-50 columns (GE Healthcare). Output cDNA (5 µL) was used as a template for 50 µL PCR reactions (Q5 Hot-start polymerase, NEB) with primers made to amplify nt 403-780 of SLNCR1 and add adapter sequences [1x Q5 reaction buffer, 250 nM each primer, 200 µM dNTPs, 3% DMSO, 0.02 Units/µL Q5 Hot-start polymerase]. PCR proceeded in a touchdown format: 98 °C for 2 minutes, 20 cycles of [98 °C for 10 s , 68 °C (decreasing by 1 °C each cycle until reaching 63 °C) for 30 s , 72 °C 30 s], and 72 °C for 2 min. Step 1 PCR products were purified using a 0.8x ratio of Agencourt AMPure XP beads (Beckman Coulter) and eluted in 20 µL of nuclease-free water. Purified PCR products (3 ng) were used as a template in 50 µL of PCR meant to add multiplex indices and remaining sequence necessary for Illumina sequencing [1xQ5 reaction buffer, 500 nM each index primer, 200 µM dNTPs, 3% DMSO, 0.02 Units/µL Q5 Hot-start polymerase]. Step 2 PCR proceeded as follows: 98 °C for 2 minutes, 10 cycles of [98 °C for 10 s , 66 °C for 30 s , 72 °C 20 s], and 72 °C for 2 min. Step 2 PCR products were purified using a 0.8x ratio of Agencourt AMPure XP beads (Beckman Coulter) and eluted in 20 µL of nuclease-free water.  [U1 snRNA MaP]  The procedure for U1 snRNA MaP RT was similar to that of SLNCR MaP with minor modifications: (1) U1-specific primers were used for RT and step 1 PCR, (2) During step 1 PCR, the touchdown protocol began with an annealing temperature of 72 °C and only decreased to 64 °C, and (3) Step 1 PCR included 500 nM of each primer, no DMSO, and only 25 µL of total volume.
attributes.tag: GEO Accession
attributes.value: GSM4152109