GSM3927363: wild-type galactose 15 mins; Saccharomyces cerevisiae BY4741; OTHER
1 g of yeast grindate was resuspended in Lysis buffer A (20 mM HEPES (pH 7.4), 110 mM KOAc, 0.5% Triton X-100, 0.1% Tween 20, 10 mM MnCl2, 1x proteinaseinhibitors (complete, EDTA-free (Roche)), 50 U/ml SUPERase.In (Invitrogen)). Resuspended grindate was incubated (4°C, 20 min) with 660 U of DNase I (Promega). Insoluble cell debris was pelleted by spinning (16,000 g, 4°C, 10 min). Supernatantswere combined (a 20 μL input sample was taken and combined with 20 μL 2 × SDS loading buffer (80 mM Tris-HCl (pH 6.8), 200 mM DTT, 3.2% SDS, 0.1% Bromophenol Blue, 1.6% glycerol)) and added to 0.5 mL of anti-FLAG M2 affinity agarose beads (Sigma) pre-washed with 2 × 10 mL Lysis buffer A (without SUPERase.In). Beads and supernatant were incubated (4°C, 2.5 h) on a nutator, before spinning (1,000 g, 4°C, 2 min). A 20 μL unbound sample was taken and combined with 20 μL 2 × SDS loading buffer. Beads were washed four times with 10 mL Wash buffer A (20 mM HEPES (pH 7.4), 110 mM KOAc, 0.5% Triton X-100, 0.1% Tween 20, 1 mM EDTA). Beads were incubated (30 min, 4°C) twice with 300 μL of Elution buffer (Lysis buffer A with 1 mg/mL 3 × FLAG peptide (Sigma)). The elution supernatants were combined (a 20 μL elute sample was taken and combined with 20 μL 2 × SDS loading buffer) and RNAs were extracted using a miRNeasy kit (QIAGEN) according to the manufacturer's instructions. For the sequential IP procedure, the elution supernatants from two IPs were combined giving a total of 1200 ul and added to 15 ul anti-HA dynabeads (Pierce) pre-washed with 2 × 1 mL Lysis buffer A. Beads and supernatant were incubated (4°C, 2.5 h) on a nutator. A 20 μL secondary unbound sample was taken and combined with 20 μL 2 × SDS loading buffer. Beads were washed five times with 1 mL Wash buffer A. Beads were resuspended in 100 uL Lysis buffer A (a 5 uL secondary elute sample was taken and combined with 15 uL Lysis buffer A and 20 uL 2 × SDS loading buffer) and RNAs were extracted using a miRNeasy kit (QIAGEN) according to the manufacturer's instructions. Samples were submitted to Harvard Biopolymers facility. They determined DNA quality and quantity using the TapeStation (Agilent) and qPCR and then sequenced samples on an Illumina HiSeq 2000 machine. 50nt were sequenced from one end using the sequencing primer. 
GEO
GSM3927363
GEO Accession
GSM3927363