GSM6008230: InputSESV5ChIPSeqS2; Homo sapiens; ChIP-Seq
GSM6008230
For RNA-seq RNA was extracted using the TRI Reagent isolation system (Sigma-Aldrich) according to the manufacturer's instructions.RNA libraries were generated starting from 1 mg of total RNA (deriving from U87, SNB19 and murine hippocampi) which quality was assessed by using a TapeStation instrument (Agilent). ATAC-seq was performed using 50000 cells for each experimental condition (SNB19 infected with SOX2V5 or SESV5), 2 technical replicates each. Briefly, cells were detached, counted, collected, and then washed by performing a centrifuge at 500 rcf for 5' at 4° in PBS. Subsequently cells were gently lysed by suspending them in 50 ul of ATAC-RSB (Corces et al., 2017) supplemented with 0.1% NP-40, 0.1% Tween-20 and 0.01% digitonin for 5' on ice. After the lysis cells were washed using 1ml of ATAC-RSB with Tween-20, but no NP-40 or digitonin. Transposition was then performed using Tn5 trasposase and buffer from Illumina (cod. 20034197). for meDiP 1μg of purified genomic DNA (gDNA) was used with qiAMP DNA mini kit (Qiagen, cat. 51304). In brief, for methylated DNA immune precipitation and purification MagMeDIPseq kit was used (Diagenode, cod. C02010040). First, gDNA was sonicated to obtain a fragment size between 150- 300 bp, then, it was de-naturated to ssDNA and immune-precipitated using α-metyl-cytosine antibody provided by the kit. The next day, immunoprecipitated DNA and Input were purified and eluted. Chromatins were isolated from SNB19. Cells were plated in adherent condition using Matrigel coated 15mm plates at a density of 6x10^6 per plate. When the plates reached 90% of confluence, cells were fixed adding formaldehyde directly to the cell culture media to reach a final concentration of 1%, then were incubated for 10 min at RT. The reaction was quenched adding glycine to a final concentration of 125 mM and incubated 5 min at RT. Medium was then removed and cells were washed 3 times with cold, sterile PBS + protease inhibitors, then cells were gently scraped and collected for centrifugation at 4°C for 50 at 1200 rpm. ChIP experiments were performed as previously described (Sessa et al. 2019). Briefly, collected cell pellets were lysed in lysis buffer (50 mM Tris-HCl pH 8, 0.1% SDS, 10 mM EDTA pH 8, 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma # P7626), protease inhibitor cocktail (Roche #04693159001) and chromatin was sonicated with a Branson D250 sonifier (4 cycles of 30 s, 20% amplitude), to reach an average fragment size of 0.1–0.5 kb. Following quantification, 100 mg of sonicated chromatin were used in each immunoprecipitation and incubated O/N at 4° C with 4 mg of V5 antibody (mouse, 1:5, ThermoFisher Scientific, R96025). RNA was processed according to the TruSeq Stranded mRNA Library Prep Kit protocol. ChIP-seq libraries were produced using 5 ng of each immunoprecipitated and purified DNA. End  repair of DNA fragments was achieved by sequential 15 min incubations at 12°C and 25°C with 0.15  U/ml T4 PNK (NEB #M0201L), 0.04 U/ml T4 POL (NEB #M0203L) and 0.1 mM dNTPs (NEB  #N0446S). A-base addition was performed by an incubation with 0.25 U/ml of Klenow fragment  (NEB # M0212L) and 167 mM dATP (NEB N0440S) for 30 min at 30°C. Adaptor ligation was  achieved by using the Quick ligation kit (NEB #M2200L) and performing an incubation of 15 min at  25°C. DNA fragments were finally amplified for 14 cycles, by using the PfuUltra II Fusion HS DNA  Pol kit (Agilent #600674). DNA purification steps after each enzymatic reaction were performed  using Agencourt AMPure XP SPRI beads (Beckman #A63882). MeDIP immunoprecipitated DNA and Input were purified and eluted. Library preparation was performed using NEBNext Ultra II kit for Illumina (cod. E7645), following  manufacturer instruction. ATAC-seq DNA After transposition was purified and libraries were amplified for the required amount of cycles  as previously described (Buenrostro et al., 2013).