GSM2797362: Mock treated sample, activated B cell, biological replicate 2; Mus musculus; OTHER
10^6 cells were washed twice with PBS1X, and incubated with 380uL of HLB buffer to strip the cytoplasmic membrane. Cells were incubated for 10 minutes with occasional flicking, then briefly vortexed, and spun at 1000G/4C/3 minutes. Nuclei were then washed three times with 1mL of HLB buffer, with spinning at 300G/4C/3 minutes. 4mL of TE low EDTA were added and 12.5uL of Proteinase K (20mG/mL). Then, 104uL of SDS 20% were added and the tubes were flipped a few times to allow homogenization. An overnight digestion at 37C allowed a complete lysis of the nuclei and subsequent release of genomic DNA. The next day, a Phenol-Chloroform extraction was performed with phase-lock tubes (5-prime). DNA was dried for 5 minutes at room temperature, resuspended in Tris-HCl 10mM pH8.0, and set to a concentration of 111.11nG/uL. Aliquots were flash-frozen in liquid nitrogen, and stored at -80C. 2.2uL of turbo DNA free buffer 10x were added to 2uG of nucleic acids in 18uL of Tris-HCl 10mM pH8.0. The reaction was spiked with 20pG of spike-in controls in 0.5uL (pool of 3ssRNAs and 1 DNA:RNA hybrid). The reaction was further divided in two x 10uL: tube “-“, and tube “+”. In tube -, DNA was incubated with 0.5uL of H2O, and in tube + with 0.5uL of RNaseH (2u/uL, ThermoFisher), at 37C. After 45 minutes, another 0.5uL of H2O or RNase H were added, and a second incubation of 45 minutes at 37C was performed. 11.25uL of Turbo DNA free buffer 1x containing 1.25uL of DNase mix were added, followed by an incubation at 37C for 45 minutes. Another 1.25uL of DNase Mix was added and the mixture incubated at 37C for 45 minutes. In order to inactivate the DNase, 0.2 volume of inactivation reagent was added and incubated for 5 minutes at room temperature, with occasional vortexings. The mixture was spun at maximum speed for 2 minutes at room temperature, and the supernatant was harvested, and heated at 65C for 10 minutes, then placed on ice. Finally, RNA was purified using RNA clean and concentrator (Zymo), eluted in 2x8uL of H2O, and used for subsequent sequencing experiment. RNAs were speedvac’d to have a total of 9uL, then 1 uL of fragmentation buffer (ThermoFisher) was added, and the mixture incubated at 70C for 2.30’. Next, 1uL of stop solution + 40uL of TT buffer were quickly added and RNA fragments were purified with RNA clean and concentrator (Zymo), and eluted in 2x6uL of H2O. RNA was quantified with Qubit 2.0 (ThermoFisher) and solution was speedvac’d to a total of 5uL. Approximatively 30nG were used for library, using the TruSeq Stranded mRNA Sample Preparation kit from Illumina, starting with incubation of RFP on page 20 (catalog #15031047, RevE), and following the manufacturer’s instructions, with the exception of the amplification step. For PCR amplification, KAPA HiFi Hotstart real-time kit was used (Kapa Biosystems). Briefly, 20uL of purified dsDNA was incubated with 5uL of PCR primer cocktail (ref 15031748, Illumina), and 25uL of 2x Master Mix from KAPA HiFi (Kapa Biosystems). Standards were also added. DNA was amplified following these conditions: 98C/45 seconds, (98C/15 seconds, 60C/30 seconds, 72C/40 seconds) x real-time. A final amplification of 72C for 2 minutes was performed, and library was purified using AMPure XP beads from Agencourt. DNA was eluted in a LoBind tube (Eppendorf) in 15uL of Tris-HCl 10mM pH8.0, and 0.05% tween was added. Library was quantified using Qubit 2.0 (ThermoFisher) and fragment sizes by Bioanalyzer (Agilent). Library was sequenced on a HiSeq instrument (Illumina).
GEO Accession
GSM2797362