title: GSM4432865: Experimental Sample 4; Gallus gallus; RNA-Seq
library_construction_protocol: HH Stage 12-13 chick embryos were electroporated with either pCIG-PtcDLoop2, which also expresses GFP, or with pMES, which expresses GFP alone. Embryos were incubated for 14 hours (until approximately HH St. 16), harvested and transferred into culture dishes containing chilled 1xPBS. Each embryo was dissected on a silicone plate using a microscalpel (Feather) to cut out the desired area (from just posterior to the heart to the posterior end of the embryo, including both sides of the embryo). The dissected tissue was transferred into chilled DMEM in a small tissue culture plate. Each sample was roughly chopped into several small pieces (4-5) and transferred, with minimal DMEM, into a 50 ml tube, containing 5ml Trypsin (0.25%, Biological Industries), on ice. Trypsin treatment was performed at 370C first in a water bath for 10 minutes and then 20 minutes in a rotary shaker (80 RPM). The reaction was stopped by adding 1ml chilled FBS. A 5ml tip was used to triturate the sample; when no visible tissue chunks were seen the sample was filtered first through a 70 μM cell strainer and then through a 40μM strainer into a 50ml tube, and then pelleted in a swinging bucket rotor, at 500G for 7 minutes. The supernatant was carefully aspirated and each sample was resuspended in 750 μl of FACS solution (1xPBS, 2% FBS, 2mM EDTA pH8) and held on ice until the FACS procedure was started.  Each embryo sample was sorted independently on a FACSAria™ IIIu (BD Biosciences) operating with BD FACSDiva 7.0 (build 2012.09.24.19.43) software, using a 561nm laser, nozzle size 100mm and pressure 4.4. Five PtcDLoop2 and five pMES electroporated embryos were sorted. Cells were determined based on FSC/SSC plot with default threshold settings. Two sequential hierarchical doublet discriminating schemes based on FSC-W/FSC-H and SSC-W/SSC-H were followed. A non electroporated eGFP negative control sample was used to determined the boundaries of positive and negative eGFP global gates. Positive cells represented approximately 0.5% of the pre-sort population for each sample. Sorted cells were collected into a 1.5ml Eppendorf tube containing 500 ml 1xPBS and 2% FBS. Tubes were centrifuged at 500g for 5 min at 40C, supernatant was removed carefully, and the pellet (containing about 500-3000 cells) was suspended in 10.5 ml of Single Cell Lysis Buffer (Clontech), incubated for 5 min at RT, pipetted to resuspend, and kept at -800C. 10 samples of 322-3100 lysed cells each were amplified separately using SMARTSeq Ultra Low Input RNA V4 kit from Clontech (cat# 634890). After the amplification step (15 cycles), the amplicons were fragmented to fragment sizes of about 300bp using Covaris E220 (Covaris,Inc.,Woburn, MA, USA). The amplified fragments were processed to libraries according to the Chip-seq protocol previously described (Blecher-Gonen et al., 2013). Libraries were evaluated by Qubit and TapeStation. Sequencing libraries were constructed with barcodes to allow multiplexing of 10 samples in one lane. Between 19-24 million single-end 60-bp reads were sequenced per sample on an Illumina HiSeq 2500 V4 instrument.
attributes.tag: GEO Accession
attributes.value: GSM4432865