title: GSM6121360: NDRG1-EGFP LMNA KO [KO10424]; Mus musculus; ATAC-seq
library_construction_protocol: Sorting protocol: Oligodendrocytes were sorted from P8 weeks NDRG1_EGFP-LMNA WT and KO brains, using fluorescence-activated cell sorting as described (Moyon et al., 2021). Tissue was dissected in HBSS 1X (HBSS 10X (Invitrogen), 0.01 M HEPES buffer, 0.75% sodium bicarbonate (Invitrogen), and 1% penicillin/streptomycin) and mechanically dissociated. After an enzymatic dissociation step using papain (30 μg/ml in DMEM-Glutamax, with 0.24 μg/ml L-cysteine and 40 μg/ml DNase I), samples were layered over a pre-formed Percoll density gradient and then centrifuged for 15 min. Cells were then collected and stained with propidium iodide (PI) for 2 min at room temperature (RT) to exclude dead cells. In a second step, GFP-positive and PI-negative cells were sorted by fluorescence-activated cell sorting (FACS; Aria, Becton Dickinson) and collected in pure fetal bovine serum. ATAC-seq reactions were performed using an established protocol (Buenrostro et al. 2013). Briefly, each sample consisted of 65000 oligodendrocyte cells sorted from the brain of NDRG1-EGFP LMNAWT or NDRG1-EGFP LMNAKO. To each cell pellet, added 25 ul of TD buffer ( 2X reaction buffer from Nextera Kit); 2.5 ul of TDE1 (Nextera TN5 Transposase from Nextera Kit) and 22.5 ul of Nuclease-free H2O. Keep at 37 degrees in gentle mixing for 30 minutes. DNA elution using Qiagen mini-elution kit. To each 10 ul of transposed DNA, 10 ul of nuclease free H2O, 2.5 ul of 25uM PCR Primer 1, 2.5 ul od 25uM Barcoded PCR Primer 2 and 25ul of NEBNext High Fidelity 2x PCR Master mix, were added. Libraries were amplified using the Nextera index kit (Illumina Cat #FC-121-1011), under the following cycling conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by thermocycling at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute for a total of 5 cycles. To avoid saturation due to over-amplification, a 10% aliquot of this reaction was then removed and subjected to qPCR for 20 cycles to calculate the optimal number of cycles needed for the remaining 90%. The additional number of cycles was determined as follows: (1) Plot linear Rn vs. Cycle (2) Calculate the # of cycles that corresponded to 1⁄4 of maximum fluorescent intensity. In general, we found adding 4 cycles to this estimate yielded optimal ATACseq libraries, as determined by analysis on Agilent D5000 screen tapes (Agilent Technologies Cat#5067-5588 and Cat#5067-5589). Libraries were amplified for a total of 13–15 cycles. Following PCR, ATACseq libraries were resolved on 2% agarose gels and fragments ranging in size from 100bp-1.5Kbp were excised and purified (Qiagen Minelute Gel Extraction Kit – Qiagen Cat#28604). Libraries were quantified by quantitative PCR (KAPA Biosystems Cat#KK4873) prior to sequencing. Libraries were sequenced on Hi-Seq2500 (Illumina) obtaining 2x50 paired-end reads.