GSM1311889: lysate_adult_testis_rep1_smallRNA; Mus musculus; RNA-Seq
Chromatoid body was purified according (Meikar et al. 2010 Exp Cell Res). Briefly after dissection seminiferouse tubels were dissociated with collagenese to the cell suspension.  The cells were crosslinked with formaledehyed, resuspend in RIPA buffer and sonicated.  The resulting lysates were centrifuged and supernatant or pellet1 were collected. The pellet1 was resuspended in RIPA buffer, sonicated again and filtrated through 5ul filters.  After centrifugation pellet2 was resuspended in RIPA buffer and incubated with Dynabeads-IgG (lnvitrogen) bounded either with rabbit MVH, IgG or ACR antibodies. After washes the IP complexes were reversed cross-links in RIPA and RNA was extracted with Trizol reagent (Invitrogen). Total RNA prepared with Trizol reagent (Invitrogen) was size selected (19-33nt) on 15% vertical polyacrylamide/urea gel. small RNA libraries were prepared according (Malone et al. Cold Spring Harb Protoc. 2012 ). Size selected RNA was ligated with 3' adaptor Modban oligonucleotide with mutant T4 RNA ligase 2, truncated (home made). The ligated products were sized fractionated on 15% vertical polyacrylamide/urea gel and ligated with 5’ adaptor with T4 RNA ligase (Ambion). Then ligated products were separated on 15% vertical polyacrylamide/urea gel and used for reverse transcription with random hexamers and Superscript lll reverse transcriptase (Invitrogen). cDNA was amplified using Illimina primers and used for following sequencing on IIllimina platphorms.
GEO Accession
GSM1311889