GSM1531844: 6F3H-AGO2_1_input; Colletotrichum higginsianum IMI 349063; RIP-Seq
1 gram of mycelia tissue was used for preparation of total and IP RNA fractions.   Small RNAs were isolated from Total RNA using size fractionantion following the protocol described in Gilbert et al., (2014, Preparation of Multiplexed Small RNA Libraries from Plants. Bio-protocol 4(20): e1275).   Immunoprecipitated small RNAs were prepared by treating clarified lysates with 4 µg/ml of Monoclonal ANTI-FLAG M2 Antibody (Sigma) for 3 h at 4ºC, followed by the addition of 100 µl Protein-G agarose beads (Roche) per milliliter of extract for 30 min at 4ºC. The entire small RNA fraction isolated from the immunoprecipation was used as input into the small RNA library protocol. Small RNA libraries were prepared following the detailed protocol described in Gilbert et al., (2014, Preparation of Multiplexed Small RNA Libraries from Plants. Bio-protocol 4(20): e1275). Modifications: 5 µl of size fractionated small RNAs were used as starting material from input fractions and the entire immunoprecipitated fraction was used for the IP libraries; 3' ligation was performed for 2 hr.
GEO Accession
GSM1531844