title: Time-course enhancer profiling of a human neuronal cell line
design: Time-course enhancer profiling of a human neuronal cell line
library_construction_protocol: About 1.5 x 10^7 cells/sample were collected at d1, d3 and d6 of differentiation. Prior collection, cells were washed twice with PBS, detached using TrypLE Express enzyme (Thermo Fisher Scientific 12605010), centrifuged at 200g for 5 minutes to remove the supernatant and pellets were flash-frozen. To lysate the cell pellet, 1500 μl of Cell Lysis Buffer composed by nuclei EZ lysis buffer (Sigma-Aldrich NUC101) supplemented with α-amanitin (FUJIFILM Wako Pure Chemical 010-22961; 25 μM), cOmplete Protease Inhibitor Cocktail (Roche 4693116001; 1x), SUPERaseIN RNase Inhibitor (Invitrogen AM2694; 20 U) and SDS (0.1%) was used. A 100 μl aliquot was taken and mixed with 700 μl of QIAzol lysis reagent (Qiagen 79306) for subsequent total RNA extraction for CAGE sequencing. The remaining cell lysate was kept in ice for 10 minutes, centrifuged at 2000g for 5 minutes at 4°C to remove the supernatant and pellet washed with 600 μl of Cell Lysys Buffer and centrifuged again at 2000g for 5 minutes at 4°C to completely remove the cytoplasmic RNA from the supernatant. Pellet was then resuspended in 200 μl of Nuclei Lysis Buffer in RNase-free water (1% NP-40, 20 mM HEPES, 300 mM NaCl, 2 M Urea, 0.2 mM EDTA, 1 mM DTT) supplemented with α-amanitin (25 μM), cOmplete Protease Inhibitor (1x), SUPERaseIN (20 U) and Empigen BB detergent (Sigma-Aldrich 30326; 0.1%), set on ice for 10 minutes and centrifuged at 3000g for 4 minutes at 4°C to remove the supernatant containing the nucleoplasmic fraction. Pellet was now representing the chromatin fraction only and was washed with 100 μl Nuclei Lysis Buffer and centrifuged at 3000g for 4 minutes at 4°C to further remove nucleoplasmic RNA. Pellet was treated with 50 μl of DNase I (Invitrogen AM2222) in RNase-free water solution (1x DNase I buffer, 50 U DNase enzyme) supplemented with SUPERaseIN (20 U) for 30 minutes at 37°C, pipetting every 5 minutes to dissolve the pellet. To isolate nascent-RNAs for NET-CAGE sequencing, 700 μl of QIAzol was added to the solution. Both nascent and total RNAs were first extracted through phenol/chloroform phases separation and then miRNeasy Mini Kit (Qiagen 217004) was used following manufacturer's instructions, including the optional on-column DNase digestion step (Qiagen 79254). RNA integrity was assessed using 2100 Bioanalyzer instrument (Agilent G2939BA). CAGE cDNA libraries were constructed following the no-amplification non-tagging CAGE libraries for Illumina next-generation sequencers (nAnT-iCAGE) protocol, while NET-CAGE cDNA libraries were generated using TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat Set B (Illumina 20020612).