title: GSM5098326: MNasecontrol_N2_muscle [wt_MNAseControl_a]; Caenorhabditis elegans; OTHER
library_construction_protocol: For ChIC-seq, filtered cells were pelleted, resuspended in 1 ml of M9, then fixed by slowly adding 2.33 mL of -20oC EtOH (100%) while vortexing cells. Cells were left at -20oC for 1h, centrifuged, and washed with wash buffer 1 (WB1) (20 mM HEPES pH 7.5, 150 mM NaCl, 0.46 mM spermidine, 0.05% Tween-20, 1x cOmplete protease inhibitor, 2 mM EDTA). Washed cells were resuspended in WB1 + 10% DMSO and stored at -80oC until ready to process. Cells were thawed on ice (all subsequent steps on ice), and washed 1x with WB1. Cells were then counted and diluted in WB1 to ~500K cells/200 µL. For each antibody and mutant, 200 µL of cells were transferred to 0.5 mL low-bind tubes (Eppendorf). 200 µL of primary antibodies, diluted to 2x in WB1, were then added cells and incubated on rotator overnight at 4oC. (Antibodies used: H3K9me3 (1:2000, MABI0318 (MBL; Kimura et al., 2008), H3K9me2 (1:5000, MABI0317 (MBL; Kimura et al., 2008) and H3K27ac (1:5000, ab177178)). Cells were washed 1x with wash buffer 2 (WB2, WB1 no EDTA), then resuspended in 500 µL of WB2 containing ProteinA-MNase (1 ng/mL - recombinant) and 5 µg/mL Hoechst 33258, and incubated on rotator 1 hr at 4oC. Cells were then washed 2x with 500 µL of WB2, then filtered into sorting tubes with ~500 µL WB2 final volume. For each sample, ~5K cells were sorted into 5 µL of wash buffer 3 (WB3, WB1 no EDTA and no protease inhibitor). Note: Hoechst was used as a sorting marker for gating fixed/lysed cells. After sorting, MNase was activated, by adding 10 µL WB3 containing 4 mM CaCl2, for 30 min on ice. The reaction was then stopped with the addition of 20 µL Stop solution (40 mM EGTA, 1.5% NP40, 2 mg/mL Proteinase K), then proteins were digested (65oC for 6h, 80oC for 20 mins, hold at 4oC). DNA fragments were blunt ended by adding 30 µL blunt end mix (2.5 U Klenow large fragment, 5 U T4-PNK, 0.4 mM dNTPs, 2.3 mM ATP, 1.7 mM MgCl2, 7 µL PNK buffer 10X, 0.24 ng/ml BSA, 2,5% PEG8000) and incubating for 30 min at 37oC followed by 20 min at 75oC for enzyme inactivation. Fragments were A-tailed by adding 30 µL tail mix (1 U AmpliTaq 360, 0.7 mM dATP, 167 mM KCL, 0.1 ng/ml BSA, 2.5% PEG8000) and incubation at 72oC for 15 min. fragments were further ligated to T-Tail containing adaptors with the sequence: forward strand: GGTGATGCCGGTAATACGACTCACTATAGG GAGTTCTACAGTCCGACGATCNNNACACACTAT, reverse strand: /5Phos/TAGTGTGTNNN GATCGTCGGACTGTAGAACTCCCTATAGTGAGTCGTATTACCGGCGAGCTT. The first 6 italic basepairs form a fork, the underlined basepairs afterwards a T7 promotor, the following underlined bases are the binding site (RA5) for the TruSeq Small RNA indexing primers (RPIx), the underlined 3 random base pairs represent the unique molecular identifier. Ligation was performed by adding 40 µL ligation mix (1.25 µM adaptor, 2000 U T4 ligase, 17.5 mM MgCl2, 52.5 mM Tris pH 7.5, 26.25 mM DTT, 1.75 mM ATP, 0.1 ng/ml BSA, 2.5% PEG8000) and incubating for 20 min at 4oC followed by 16 hrs at 16oC. Ligase was inactivated by incubation for 10 min at 65oC. Ligation reactions were cleaned with Ampure XP beads at a 0.8 beads to sample ratio and resuspended in 8 µL clean water. Cleaned fragments were amplified by adding 12 µL of MEGAscript™ T7 Transcription Kit and incubation for 12 hrs at 37oC. Template DNA is removed by addition of 2 µL TurboDNAse (IVT kit) and incubation for 15 min at 37oC. The produced RNA is further purified using RNA Clean XP beads at 0.8 beads to sample ratio, followed by RNA fragmentation for 2 min at 94oC. After another bead cleanup, 40% (5 µL) of the RNA is primed for reverse transcription by adding 1,5 µL of 3.3 mM dNTPs and 13.3 µM randomhexamer RT primer (GCCTTGGCACCCGAGAATTCCANNNNNN) and hybridizing it by incubation at 65oC for 5 min followed by direct cool down on ice. Reverse transcription is performed by further addition of 4 µL RT mix (2.5x first strand buffer, 25 mM DTT, 20 U RNAseOUT and 100 U SuperscriptII) and incubating at 25oC for 10 min followed by 1 hr at 42oC. Single stranded DNA is purified through incubation with 5 ug/25 U RNAseA for 30 min at 37oC and PCR amplification to add the Illumina smallRNA barcodes and handles. 40 µL of PCR mix (3.1x NEBNext Ultra II Q5 Master Mix (5x stock), 0.5 µM RP1 and 0.5 µM RPIx primers) was added. PCR cycles depended on the abundance of the histone modification assayed and PCR products were cleaned twice Ampure XP beads at a 0.8x bead to sample ratio. The final libraries were eluted in 7 µL Nuclease free water and abundance and quality were assessed by QUBIT and bioanalyzer.