title: GSM4403465: Input_L1_r1; Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: Packed worms were grounded under liquid nitrogen and brought to 10 ml with FA buffer (50 mM HEPES/KOH pH7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl). Next, 600 μl of 37% formaldehyde were added and allowed to crosslink for 20 mins at room temperature. Glycine (600 μl; 2.5M) was used to quench the reaction, for 2 mins, and nuclei were recovered by centrifugation (4000 rpm, 5 mins in a microfuge), washed 3 times with FA buffer by resuspending in 5 ml and spinning again. Finally, nuclei were resuspended in 1.6 ml of FA buffer, split into two Eppendorf tubes and sonicated for 10 mins in a Bioruptor® Pico (Diagenode) under standard conditions (30s on, 30s off). After sonication, protein extract was quantified and approximately 2 mg of each sample were spun at full speed for 10 minutes at 4oC and the supernatants (SN) were pooled again. SN were pre-cleared with FA-equilibrated SPA/G beads (GE Healthcare) by rotating for 30 minutes at room temperature, recovered by spinning at 1200 rpm for 2 mins and used for immunoprecipitation (IP). An aliquot of 100 μl was kept as input control at this point. The rest of the sample was split into different tubes for IP with the antibodies of choice. After adding the corresponding antibody (3-10 μg), samples were incubated in a rotator overnight at 4oC and the immunocomplexes were recovered with FA buffer pre-equilibrated SPA/G beads (GE Healthcare) by rotating 2h at 4oC. Beads were pelleted (1200rpm; 2 mins) washed twice with FA buffer (5 mins each); once with FA + 0.5 M NaCl (10 mins); once with FA buffer + 1M NaCl (5 mins); once with lithium buffer (10 mins); twice with TE buffer (5 mins each) and the immunoprecipitated material was recovered by incubation in 100 μl elution buffer (Tris-EDTA; 1% SDS plus inhibitors) 1h at RT. DNA was finally purified by reverse crosslinking at 65oC overnight followed by proteinase K (0.5 or 1 mg/ml, respectively) digestions and purifying with MinElute® PCR purification kit (Qiagen). Samples were digested with 0.1% collagenase and single-cell suspension was stained as described. Different subpopulations were directly sorted in a total volume of 10  ml of reaction buffer and processed for obtaining cDNA following manufacture's protocol. cDNA amplification was performed by Long Distance PCR (LD-PCR) and the PCR-amplified cDNA purified by immobilization on AMPure XP beads (Agencourt AMPure XP kit). Samples were analyzed with Agilent High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626). Covaris shearing was used for Illumina Low input sample preparation and Double last bead purification was performed to remove fragments below 200 bp. Next, samples were used to generate an Illumina sequencing library by NEBNext Ultra protocol following kit instructions. After PCR amplification of the library, the quality was checked on a Bioanalyzer and total cDNA was sequenced using an Illumina HiSeq 2000 sequencer.
attributes.tag: GEO Accession
attributes.value: GSM4403465