title: GSM4661286: KLp_3_RRBS; Mus musculus; Bisulfite-Seq
library_construction_protocol: 24 hours after status epilepticus right hippocampi were extracted and flash frozen. Tissue was thawed in homogenization buffer and homogenized on a GentleMACS dissociator (Miltenyi). Debris was removed by density gradient centrifugation, using Debris Removal Solution (Miltenyi cat.no. 130-109-398). Nuclei were resuspended in buffer and stained with anti-NeuN-Alexa Fluor488 (Millipore cat.no. MAB377X), followed by sorting on BD FACSAria (BD Biosciences). DNA was extracted from NeuN positive nuclei with MasterPure Complete DNA and RNA Purification Kit (Epicentre cat.no. MC85200), following the DNA purification protocol for cell samples. 100ng DNA was digested with MspI (New England Biolabs cat.no. R0106S), followed by gap filling and A-tailing using Klenow fragment (NEB cat.no. M0212S) and dCTP, dGTP, and dATP (NEB cat.no. N0446S). Clean-up was performed using 3 volumes of AMPure XP beads (Beckman Coulter cat.no. A63880). 1 µL of a 1:10 dilution of adaptors from Illumina kit FC-121-3001/2 or FC-121-4001/2 was used for indexing of samples, with ligation performed by T4 ligase (NEB cat.no. M0202S). A two-sided size selection using AMPure XP beads was performed to recover ligation products between approximately 200 and 400 bp. Size selected fragments were bisulfite converted with the EpiTect Bisulfite Kit (Qiagen cat.no. 59104) using the procedure for FFPE samples and two consecutive rounds of the incubations given in the protocol. Bisulfite converted samples were amplified using TruSeq forward and reverse primers and PfuTurbo Cx hotstart DNA polymerase (Agilent cat.no. 600412), followed by AMPure XP clean-up.
attributes.tag: GEO Accession
attributes.value: GSM4661286